Variant libraries of the immunological synapse and synapse and synthesis thereof

ABSTRACT

Disclosed herein are methods for the generation of highly accurate nucleic acid libraries encoding for predetermined variants of a nucleic acid sequence. The nucleic acid sequence may encode for all or part of a reference domain of a CAR. The degree of variation may be complete, resulting in a saturated variant library, or less than complete, resulting in a non-saturating library of variants. The variant nucleic acid libraries described herein may be designed for further processing by transcription or translation. The variant nucleic acid libraries described herein may be designed to generate variant RNA, DNA and/or protein populations. Further provided herein are method for identifying variant species with increased or decreased activities, with applications in regulating biological functions and the design of therapeutics for treatment or reduction of a disease, such as cancer.

CROSS-REFERENCE

This application is a divisional of U.S. patent application Ser. No.15/921,537, filed on Mar. 14, 2018, which claims benefit of U.S.Provisional Patent Application No. 62/471,810 filed on Mar. 15, 2017,which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 9, 2018, isnamed 44854-733_401_SL.txt and is 19,476 bytes in size.

BACKGROUND

The cornerstone of synthetic biology is the design, build, and testprocess—an iterative process that requires DNA, to be made accessiblefor rapid and affordable generation and optimization of these custompathways and organisms. In the design phase, the A, C, T and Gnucleotides that constitute DNA are formulated into the various genesequences that would comprise the locus or the pathway of interest, witheach sequence variant representing a specific hypothesis that will betested. These variant gene sequences represent subsets of sequencespace, a concept that originated in evolutionary biology and pertains tothe totality of sequences that make up genes, genomes, transcriptome andproteome.

Many different variants are typically designed for eachdesign-build-test cycle to enable adequate sampling of sequence spaceand maximize the probability of an optimized design. Thoughstraightforward in concept, process bottlenecks around speed, throughputand quality of conventional synthesis methods dampen the pace at whichthis cycle advances, extending development time. The inability tosufficiently explore sequence space due to the high cost of acutelyaccurate DNA and the limited throughput of current synthesistechnologies remains the rate-limiting step.

Beginning with the build phase, two processes are noteworthy:oligonucleotide synthesis and gene synthesis. Historically, synthesis ofdifferent gene variants was accomplished through molecular cloning.While robust, this approach is not scalable. Early chemical genesynthesis efforts focused on producing a large number ofoligonucleotides with overlapping sequence homology. These were thenpooled and subjected to multiple rounds of polymerase chain reaction(PCR), enabling concatenation of the overlapping oligonucleotides into afull length double stranded gene. A number of factors hinder thismethod, including time-consuming and labor-intensive construction,requirement of high volumes of phosphoramidites, an expensive rawmaterial, and production of nanomole amounts of the final product,significantly less than required for downstream steps, and a largenumber of separate oligonucleotides required one 96 well plate to set upthe synthesis of one gene.

Synthesizing oligonucleotides on microarrays provided a significantincrease in the throughput of gene synthesis. A large number ofoligonucleotides could be synthesized on the microarray surface, thencleaved off and pooled together. Each oligonucleotide destined for aspecific gene contains a unique barcode sequence that enabled thatspecific subpopulation of oligonucleotides to be depooled and assembledinto the gene of interest. In this phase of the process, each subpool istransferred into one well in a 96 well plate, increasing throughput to96 genes. While this is two orders of magnitude higher in throughputthan the classical method, it still does not adequately support thedesign, build, test cycles that require thousands of sequences at onetime due to a lack of cost efficiency and slow turnaround times. Thus,there is a need for more efficient generation of variant sequencelibraries.

The immune system has the ability to seek and destroy harmful cells. Tcells play an important role in such a process. As such, therapiescomprising genetically modified T cells to teach the immune system torecognize and eliminate malignant cells are alternative therapies thatmay be used to treat diseases such as cancer. In particular, geneticallymodified T cells that express chimeric antigen receptors (CARs) may bedesigned to target deleterious cells, such as cancer cells. However, CARtherapies require further optimization due to their ability to causeundesirable effects such as on-target off-tumor toxicity, autoimmunity,host-graft toxicity, and sometimes death. Thus, there is a need forimproved compositions and methods for cancer therapies utilizing CARtherapies.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF SUMMARY

Provided herein are nucleic acid libraries, wherein the nucleic acidlibrary comprises at least 10,000 nucleic acids, wherein each nucleicacid encodes for a preselected variant of a reference sequence thatencodes for a chimeric antigen receptor or a functional domain thereof.Further provided herein are nucleic acid libraries, wherein thefunctional domain of the chimeric antigen receptor comprises an antigenrecognition domain, a hinge domain, a transmembrane domain, or anintracellular domain. Further provided herein are nucleic acidlibraries, wherein the library comprises at least 1,000,000 nucleicacids. Further provided herein are nucleic acid libraries, wherein eachnucleic acid is 100 bases to 2000 bases in length. Further providedherein are nucleic acid libraries, wherein each nucleic acid is attachedto a vector sequence. Further provided herein are nucleic acidlibraries, wherein the vector sequence is a viral vector sequence.

Provided herein are nucleic acid libraries, wherein the nucleic acidlibrary comprises a plurality of nucleic acids, wherein each nucleicacid encodes for a preselected variant of a reference sequence thatencodes for a chimeric antigen receptor or a functional domain thereof,wherein the plurality of nucleic acids comprises all variations for atleast two positions in the reference sequence, and wherein the at leasttwo positions encode sequences from 5 to 20 different codons. Furtherprovided herein are nucleic acid libraries, wherein the functionaldomain of the chimeric antigen receptor comprises an antigen recognitiondomain, a hinge domain, a transmembrane domain, or an intracellulardomain. Further provided herein are nucleic acid libraries, wherein theplurality of nucleic acids comprises all variations for 2, 3, 4, or 5positions in the reference sequence. Further provided herein are nucleicacid libraries, wherein the at least two positions encode sequences from10 to 20 different codons. Further provided herein are nucleic acidlibraries, wherein the at least two positions encode sequences for about10 different codons. Further provided herein are nucleic acid libraries,wherein each nucleic acid is 100 bases to 2000 bases in length.

Provided herein are oligonucleotide libraries, the oligonucleotidelibrary comprising at least 10,000 oligonucleotides, wherein eacholigonucleotide encodes for a preselected variant of a referencesequence that encodes for a region of a chimeric antigen receptor,wherein the region comprises a portion of an antigen recognition domain,a hinge domain, a transmembrane domain, or an intracellular domain.Further provided herein are oligonucleotide libraries, wherein thelibrary comprises at least 1,000,000 oligonucleotides. Further providedherein are oligonucleotide libraries, wherein each oligonucleotide is 12to 500 bases in length. Further provided herein are oligonucleotidelibraries, wherein each oligonucleotide is attached to a vectorsequence.

Provided herein are oligonucleotide libraries, wherein theoligonucleotide library comprises a plurality of oligonucleotides,wherein each oligonucleotide encodes for a preselected variant of areference sequence that encodes for a region of a chimeric antigenreceptor, wherein the region comprises a portion of an antigenrecognition domain, a hinge domain, a transmembrane domain, or anintracellular domain, wherein each oligonucleotide is at least 12 basesin length, wherein the plurality of oligonucleotides comprises allvariations for at least two positions in the reference sequence, andwherein the at least two positions encode sequences from 5 to 20different codons. Further provided herein are oligonucleotide libraries,wherein the plurality of oligonucleotides comprises all variations for2, 3, 4, or 5 positions. Further provided herein are oligonucleotidelibraries, wherein the at least two positions encode sequences from 10to 20 different codons. Further provided herein are oligonucleotidelibraries, wherein the at least two positions encode sequences for about10 different codons. Further provided herein are oligonucleotidelibraries, wherein each oligonucleotide is 12 to 500 bases in length.

Provided herein are nucleic acid libraries comprising at least 400nucleic acids, wherein a first plurality of the at least 400 nucleicacids encodes for a variant of a reference sequence that encodes for anantigen recognition domain, and wherein a second plurality of the atleast 400 nucleic acids encodes for a variant of a reference sequencethat encodes for a hinge domain, a transmembrane domain, or anintracellular domain of a chimeric antigen receptor (CAR). Furtherprovided herein are nucleic acid libraries, wherein each nucleic acid isat least 100 bases in length. Further provided herein are nucleic acidlibraries, wherein each nucleic acid is 100 to 2000 bases in length.Further provided herein are nucleic acid libraries, wherein the firstplurality of the at least 400 nucleic acids comprises all variations forat least two positions in the reference sequence. Further providedherein are nucleic acid libraries, wherein the first plurality of the atleast 400 nucleic acids comprises all variations for 2, 3, 4, or 5positions in the reference sequence. Further provided herein are nucleicacid libraries, wherein the second plurality of the at least 400 nucleicacids comprises all variations for at least two positions in thereference sequence. Further provided herein are nucleic acid libraries,wherein the second plurality of the at least 400 nucleic acids comprisesall variations for 2, 3, 4, or 5 positions in the reference sequence.Further provided herein are nucleic acid libraries, wherein the at leasttwo positions encode sequences from 5 to 20 different codons. Furtherprovided herein are nucleic acid libraries, wherein the at least twopositions encode sequences from 10 to 20 different codons. Furtherprovided herein are nucleic acid libraries, wherein the at least twopositions encode sequences for about 10 different codons.

Provided herein are methods of synthesizing a nucleic acid library,comprising: (a) providing a first set of preselected oligonucleotidesequences encoding for at least 400 sequences of a chimeric antigenreceptor gene or gene fragment, wherein each sequence comprises at leastone variation of at least two preselected codons for an amino acidresidue in an antigen recognition domain; (b) synthesizing the first setof preselected oligonucleotide sequences; and (c) screening a firstactivity for proteins encoded by the first set of oligonucleotidesequences, wherein the first activity is specificity, avidity, affinity,stability, or expression. Further provided herein are methods ofsynthesizing a nucleic acid library, wherein the antigen is a cancerantigen. Further provided herein are methods of synthesizing a nucleicacid library, wherein the cancer antigen is MAGE A3, MAGE A12, MAGE A2,MAGE A6, NY-ESO-1, or CEA. Further provided herein are methods ofsynthesizing a nucleic acid library, wherein each sequence comprises upto 100 variations at preselected codons for amino acid residues in theantigen recognition domain. Further provided herein are methods ofsynthesizing a nucleic acid library, wherein each sequence comprises upto 30 variations at preselected codons for amino acid residues in theantigen recognition domain. Further provided herein are methods ofsynthesizing a nucleic acid library further comprising (a) providing asecond set of preselected oligonucleotide sequences encoding for atleast one sequence of a chimeric antigen receptor gene or gene fragment,where each sequence comprises at least one variation at a preselectedcodon for an amino acid residue in the chimeric antigen receptor in ahinge domain, a transmembrane domain, or an intracellular domain; (b)synthesizing the second set of preselected oligonucleotide sequences;and (c) screening a second activity for proteins encoded by the secondset of oligonucleotide sequences, wherein the second activity isspecificity, avidity, affinity, stability, or expression, and whereinthe second activity is different from the first activity.

Provided herein is an oligonucleotide library, the library comprising atleast 10,000 variant oligonucleotides, wherein each variantoligonucleotide is at least 12 bases in length, wherein each variantoligonucleotide encodes for a variant of a reference sequence thatencodes for an antigen recognition domain, a hinge domain, atransmembrane domain, or an intracellular domain of a chimeric antigenreceptor, and wherein at least 80% of the variant oligonucleotides haveno errors compared to the predetermined sequences without correctingerrors. Further provided herein is an oligonucleotide library whereinthe library comprises at least 1,000,000 variant oligonucleotides.Further provided herein is an oligonucleotide library, wherein eachvariant oligonucleotide is 20 to 500 bases in length. Further providedherein is an oligonucleotide library wherein at least 90% of the variantoligonucleotides have no errors compared to the predetermined sequenceswithout correcting errors.

Provided herein is a nucleic acid library, the library comprising atleast 10,000 variant nucleic acids, wherein each variant nucleic acidencodes for a variant of a reference sequence for a chimeric antigenreceptor, wherein the variation occurs in the sequence for an antigenrecognition domain, a hinge domain, a transmembrane domain, or anintracellular domain of a chimeric antigen receptor, and wherein atleast 70% of the variant nucleic acids have no errors compared to thepredetermined sequences without correcting errors. Further providedherein is a nucleic acid library wherein the library comprises about10,000,000 variant nucleic acids. Further provided herein is a nucleicacid library wherein each variant nucleic acid is 600 to 3000 bases inlength. Further provided herein is a nucleic acid library wherein eachvariant nucleic acid is attached to a vector sequence. Further providedherein is a nucleic acid library wherein the vector sequence is a viralvector sequence.

Provided herein is a method for nucleic acid synthesis, the methodcomprising: providing predetermined sequences encoding for a pluralityof non-identical oligonucleotides, wherein each of the non-identicaloligonucleotides is at least 20 bases in length, and wherein theplurality of non-identical oligonucleotides encodes for up to 19variants for each of at least 2 codons compared to at least onereference sequence, wherein the at least one reference sequence encodesfor a region of an antigen recognition domain, a hinge domain, atransmembrane domain, or an intracellular domain of a chimeric antigenreceptor; providing a structure having a surface, wherein the surfacecomprises clusters of loci for oligonucleotide extension, each clustercomprising 50 to 500 loci; synthesizing the plurality of non-identicaloligonucleotides, wherein each of the non-identical oligonucleotidesextends from a separate locus; and performing an amplification reactionto assemble a library of variant nucleic acids, wherein theamplification reaction comprises mixing the plurality of non-identicaloligonucleotides from a single cluster with a DNA polymerase and the atleast one reference sequence. Further provided herein is a methodwherein each cluster further comprises additional oligonucleotides, andwherein the plurality of non-identical oligonucleotides and theadditional oligonucleotides within said cluster collectively encode fora single gene and variants thereof. Further provided herein is a methodwherein the surface of the structure comprises at least 6000 of theclusters. Further provided herein is a method wherein at least 80% ofthe variant nucleic acids have no errors compared to the predeterminedsequences without correcting errors. Further provided herein is a methodwherein at least 90% of the variant nucleic acids have no errorscompared to the predetermined sequences without correcting errors.Further provided herein is a method wherein a replicate of eachnon-identical oligonucleotide extends from the surface at up to 5 loci.Further provided herein is a method wherein a replicate of eachnon-identical oligonucleotide extends from the surface at up to 3 loci.Further provided herein is a method wherein the plurality ofnon-identical oligonucleotides encodes for up to 19 variants for each ofat least 3 codons compared to at least one reference sequence. Furtherprovided herein is a method further comprising expressing the variantnucleic acid library, wherein expression of the variant nucleic acidlibrary provides for a variant protein having improved affinity,improved gene expression, improved avidity, improved stability, orimproved target specificity compared to the expression product of thereference sequence.

Provided herein is a method for nucleic acid synthesis, the methodcomprising: providing predetermined sequences encoding for a pluralityof non-identical oligonucleotides, wherein each of the non-identicaloligonucleotides is at least 20 bases in length, and wherein theplurality of non-identical oligonucleotides encodes for up to 19variants for each of at least 2 codons compared to at least onereference sequence, wherein the at least one reference sequence encodesfor an epitope to a chimeric antigen receptor; providing a structurehaving a surface, wherein the surface comprises clusters of loci foroligonucleotide extension, each cluster comprising 50 to 500 loci;synthesizing the plurality of non-identical oligonucleotides, whereineach of the non-identical oligonucleotides extends from a separatelocus; and performing an amplification reaction to assemble a library ofvariant nucleic acids, wherein the amplification reaction comprisesmixing the plurality of non-identical oligonucleotides from a singlecluster with a DNA polymerase and the at least one reference sequence.Further provided herein is a method wherein the surface of the structurecomprises at least 6000 of the clusters. Further provided herein is amethod wherein at least about 80% of the variant nucleic acids have noerrors compared to the predetermined sequences without correctingerrors. Further provided herein is a method wherein at least about 90%of the variant nucleic acids have no errors compared to thepredetermined sequences without correcting errors. Further providedherein is a method wherein a replicate of each non-identicaloligonucleotide extends from the surface at up to 5 loci. Furtherprovided herein is a method wherein a replicate of each non-identicaloligonucleotide extends from the surface at up to 3 loci. Furtherprovided herein is a method wherein the plurality of non-identicaloligonucleotides encodes for up to 19 variants for each of at least 3codons compared to at least one reference sequence. Further providedherein is a method further comprising expressing the variant nucleicacid library, wherein expression of the variant nucleic acid libraryprovides for a variant peptide having improved affinity, improved geneexpression, improved avidity, improved stability, or improved targetspecificity compared to the expression product of the referencesequence. Further provided herein is a method wherein the at least onereference sequence encodes for the epitope to a chimeric antigenreceptor and also for an epitope to a surface protein expressed by anon-lymphoid cell.

Provided herein is a method for nucleic acid synthesis of nucleic acidlibraries for optimization assays, the method comprising: providing afirst set of predetermined sequences encoding for a first plurality ofnon-identical oligonucleotides, wherein each of the non-identicaloligonucleotides is at least 20 bases in length, and wherein the firstplurality of non-identical oligonucleotides encodes for up to 19variants for each of at least 3 codons compared to at least onereference sequence, wherein the at least one reference sequence encodesfor a region of an antigen recognition domain, a hinge domain, atransmembrane domain, or an intracellular domain of a chimeric antigenreceptor; providing a second set of predetermined sequences encoding fora second plurality of non-identical oligonucleotides, wherein each ofthe second non-identical oligonucleotides is at least 20 bases inlength, and wherein the second plurality of non-identicaloligonucleotides encodes for up to 19 variants for each of at least 3codons compared to at least one reference sequence, wherein the at leastone reference sequence encodes for an epitope to the chimeric antigenreceptor; providing a structure having a surface, wherein the surfacecomprises clusters of loci for oligonucleotide extension, each clustercomprising 50 to 500 loci; synthesizing both the first plurality ofnon-identical oligonucleotides and the second plurality of non-identicaloligonucleotides, wherein each non-identical oligonucleotides extendsfrom a separate locus; and performing an amplification reaction toassemble a first library of nucleic acids, wherein the amplificationreaction comprises mixing the first plurality of non-identicaloligonucleotides from a single cluster with a DNA polymerase and the atleast one reference sequence; performing an amplification reaction toassemble a second library of variant nucleic acids, wherein theamplification reaction comprises mixing the second plurality ofnon-identical oligonucleotides from a single cluster with a DNApolymerase and the at least one reference sequence. Further providedherein is a method further comprising screening purified proteinexpression products of the second library of variant nucleic acidsagainst a library of cells expressing the first library of variantnucleic acids, wherein the library of cells comprises populations ofcells, each population of cells expressing a different proteinexpression product encoded by the first library of variant nucleicacids. Further provided herein is a method wherein the at least onereference sequence for an epitope to the chimeric antigen receptor alsoencodes for an epitope to a surface protein expressed by a non-lymphoidcell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D depict a process workflow for the synthesis of variantbiological molecules incorporating a PCR mutagenesis step.

FIGS. 2A-2D depict a process workflow for the generation of a nucleicacid comprising a nucleic acid sequence which differs from a referencenucleic acid sequence at a single predetermined codon site.

FIGS. 3A-3F depict an alternative workflow for the generation of a setof nucleic acid variants from a template nucleic acid, with each variantcomprising a different nucleic acid sequence at a single codon position.Each variant nucleic acid encodes for a different amino acid at theirsingle codon position, the different codons represented by X, Y, and Z.

FIGS. 4A-4E depict a reference amino acid sequence (FIG. 4A) having anumber of amino acids, each residue indicated by a single circle, andvariant amino acid sequences (FIGS. 4B, 4C, 4D, & 4E) generated usingmethods described herein. The reference amino acid sequence and variantsequences are encoded by nucleic acids and variants thereof generated byprocesses described herein.

FIGS. 5A-5B depict a reference amino acid sequence (FIG. 5A) and alibrary of variant amino acid sequences (FIG. 5B), each variantcomprising a single residue variant (indicated by an “X”). The referenceamino acid sequence and variant sequences are encoded by nucleic acidsand variants thereof generated by processes described herein. FIG. 5Adiscloses SEQ ID NO: 42 and FIG. 5B discloses SEQ ID NOS 43-49,respectively, in order of appearance.

FIGS. 6A-6B depict a reference amino acid sequence (FIG. 6A) and alibrary of variant amino acid sequences (FIG. 6B), each variantcomprising two sites of single position variants. Each variant isindicated by differently patterned circles. The reference amino acidsequence and variant sequences are encoded by nucleic acids and variantsthereof generated by processes described herein.

FIGS. 7A-7B depict a reference amino acid sequence (FIG. 7A) and alibrary of variant amino acid sequences (FIG. 7B), each variantcomprising a stretch of amino acids (indicated by a box around thecircles), each stretch having three sites of position variants (encodingfor histidine) differing in sequence from the reference amino acidsequence. The reference amino acid sequence and variant sequences areencoded by nucleic acids and variants thereof generated by processesdescribed herein.

FIGS. 8A-8B depict a reference amino acid sequence (FIG. 8A) and alibrary of variant amino acid sequences (FIG. 8B), each variantcomprising two stretches of amino acid sequence (indicated by a boxaround the circles), each stretch having one site of single positionvariants (illustrated by the patterned circles) differing in sequencefrom reference amino acid sequence. The reference amino acid sequenceand variant sequences are encoded by nucleic acids and variants thereofgenerated by processes described herein.

FIGS. 9A-9B depict a reference amino acid sequence (FIG. 9A) and alibrary of amino acid sequence variants (FIG. 9B), each variantcomprising a stretch of amino acids (indicated by patterned circles),each stretch having a single site of multiple position variantsdiffering in sequence from the reference amino acid sequence. In thisillustration, 5 positions are varied where the first position has a50/50 K/R ratio; the second position has a 50/25/25 V/L/S ratio, thethird position has a 50/25/25 Y/R/D ratio, the fourth position has anequal ratio for all amino acids, and the fifth position has a 75/25ratio for G/P. The reference amino acid sequence and variant sequencesare encoded by nucleic acids and variants thereof generated by processesdescribed herein.

FIGS. 10A-10C illustrate generations of chimeric antigen receptors(CARs). CARs comprise an extracellular single chain variable fragment(scFv), a hinge domain (H), a transmembrane domain (TM), and anintracellular domain. An intracellular domain of a first generation CARcomprises CDζ alone (FIG. 10A). An intracellular domain of a secondgeneration CAR comprises a costimulatory domain and CD3ζ (FIG. 10B). Anintracellular domain of a third generation CAR comprises twocostimulatory domains and CD3ζ (FIG. 10C). In FIGS. 10D-10E, various CARbinding arrangements are illustrated.

FIG. 11 depicts an exemplary number of variants produced byinterchanging sections of two expression cassettes (e.g., promotors,open reading frames, and terminators) to generate a variant library ofexpression cassettes.

FIG. 12 presents a diagram of steps demonstrating an exemplary processworkflow for gene synthesis as disclosed herein.

FIG. 13 illustrates an example of a computer system.

FIG. 14 is a block diagram illustrating an architecture of a computersystem.

FIG. 15 is a diagram demonstrating a network configured to incorporate aplurality of computer systems, a plurality of cell phones and personaldata assistants, and Network Attached Storage (NAS).

FIG. 16 is a block diagram of a multiprocessor computer system using ashared virtual address memory space.

FIG. 17 depicts a BioAnalyzer plot of PCR reaction products resolved bygel electrophoresis.

FIG. 18 depicts an electropherogram showing 96 sets of PCR products,each set of PCR products differing in sequence from a wild-type templatenucleic acid at a single codon position, where the single codon positionof each set is located at a different site in the wild-type templatenucleic acid sequence. Each set of PCR products comprises 19 variantnucleic acids, each variant encoding for a different amino acid at theirsingle codon position.

DETAILED DESCRIPTION

The present disclosure employs, unless otherwise indicated, conventionalmolecular biology techniques, which are within the skill of the art.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art.

Definitions

Throughout this disclosure, numerical features are presented in a rangeformat. It should be understood that the description in range format ismerely for convenience and brevity and should not be construed as aninflexible limitation on the scope of any embodiments. Accordingly, thedescription of a range should be considered to have specificallydisclosed all the possible subranges as well as individual numericalvalues within that range to the tenth of the unit of the lower limitunless the context clearly dictates otherwise. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual valueswithin that range, for example, 1.1, 2, 2.3, 5, and 5.9. This appliesregardless of the breadth of the range. The upper and lower limits ofthese intervening ranges may independently be included in the smallerranges, and are also encompassed within the disclosure, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the disclosure, unless thecontext clearly dictates otherwise.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of any embodiment.As used herein, the singular forms “a,” “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof. As used herein, the term “and/or”includes any and all combinations of one or more of the associatedlisted items.

Unless specifically stated or obvious from context, as used herein, theterm “about” in reference to a number or range of numbers is understoodto mean the stated number and numbers +/−10% thereof, or 10% below thelower listed limit and 10% above the higher listed limit for the valueslisted for a range.

The term “nucleic acid” encompasses double- or triple-stranded nucleicacids, as well as single-stranded molecules. In double- ortriple-stranded nucleic acids, the nucleic acid strands need not becoextensive (i.e., a double-stranded nucleic acid need not bedouble-stranded along the entire length of both strands). Nucleic acidsequences, when provided, are listed in the 5′ to 3′ direction, unlessstated otherwise. Methods described herein provide for the generation ofisolated nucleic acids. Methods described herein additionally providefor the generation of isolated and purified nucleic acids.

As used herein, the terms “preselected sequence,” “predefined sequence”or “predetermined sequence” are used interchangeably. The terms meanthat the sequence of the polymer is known and chosen before synthesis orassembly of the polymer. In particular, various aspects of the inventionare described herein primarily with regard to the preparation of nucleicacids molecules, the sequence of the oligonucleotide or polynucleotidebeing known and chosen before the synthesis or assembly of the nucleicacid molecules.

Provided herein are methods and compositions for production of synthetic(i.e. de novo synthesized or chemically synthesized) polynucleotides.The term oligonucleotide, oligo, oligonucleic acid, and polynucleotideare defined to be synonymous throughout. Libraries of synthesizedpolynucleotides described herein may comprise a plurality ofpolynucleotides collectively encoding for one or more genes or genefragments. In some instances, the polynucleotide library comprisescoding or non-coding sequences. In some instances, the polynucleotidelibrary encodes for a plurality of cDNA sequences. Reference genesequences from which the cDNA sequences are based may contain introns,whereas cDNA sequences exclude introns. Polynucleotides described hereinmay encode for genes or gene fragments from an organism. Exemplaryorganisms include, without limitation, prokaryotes (e.g., bacteria) andeukaryotes (e.g., mice, rabbits, humans, and non-human primates). Insome instances, the polynucleotide library comprises one or morepolynucleotides, each of the one or more polynucleotides encodingsequences for multiple exons. Each polynucleotide within a librarydescribed herein may encode a different sequence, i.e., non-identicalsequence. In some instances, each polynucleotide within a librarydescribed herein comprises at least one portion that is complementary toa sequence of another polynucleotide within the library. Polynucleotidesequences described herein may, unless stated otherwise, comprise DNA orRNA.

Provided herein are methods and compositions for production of synthetic(i.e. de novo synthesized) genes. Libraries comprising synthetic genesmay be constructed by a variety of methods described in further detailelsewhere herein, such as PCA, non-PCA gene assembly methods orhierarchical gene assembly, combining (“stitching”) two or moredouble-stranded oligonucleotides to produce larger DNA units (i.e., achassis). Libraries of large constructs may involve oligonucleotidesthat are at least 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40,50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500 kb longor longer. The large constructs may be bounded by an independentlyselected upper limit of about 5000, 10000, 20000 or 50000 base pairs.The synthesis of any number of polypeptide-segment encoding nucleotidesequences, including sequences encoding non-ribosomal peptides (NRPs),sequences encoding non-ribosomal peptide-synthetase (NRPS) modules andsynthetic variants, polypeptide segments of other modular proteins, suchas antibodies, polypeptide segments from other protein families,including non-coding DNA or RNA, such as regulatory sequences e.g.promoters, transcription factors, enhancers, siRNA, shRNA, RNAi, miRNA,small nucleolar RNA derived from microRNA, or any functional orstructural DNA or RNA unit of interest. The following are non-limitingexamples of polynucleotides: coding or non-coding regions of a gene orgene fragment, intergenic DNA, loci (locus) defined from linkageanalysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomalRNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA(miRNA), small nucleolar RNA, ribozymes, complementary DNA (cDNA), whichis a DNA representation of mRNA, usually obtained by reversetranscription of messenger RNA (mRNA) or by amplification; DNA moleculesproduced synthetically or by amplification, genomic DNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probes,and primers. cDNA encoding for a gene or gene fragment referred toherein, may comprise at least one region encoding for exon sequence(s)without an intervening intron sequence found in the correspondinggenomic sequence. Alternatively, the corresponding genomic sequence to acDNA may lack an intron sequence in the first place.

Chimeric Antigen Receptor

Chimeric antigen receptors (CARs) are generally engineered receptorsthat recognize a particular antigen such as an antigen whose epitope isunique to cancer cells. Referring to FIGS. 10A-10C, CARs comprise anantigen recognition domain derived from a single chain antibody (scFv),a hinge domain (H) or spacer, a transmembrane domain (TM) that anchorsthe CAR to the plasma membrane, and an intracellular domain thatmediates T cell activation. An intracellular domain of a firstgeneration CAR comprises CD3ζ (FIG. 10A). An intracellular domain of asecond generation CAR comprises a costimulatory domain and CD3ζ (FIG.10B). An intracellular domain of a third generation CAR comprises twocostimulatory domains and CD3ζ (FIG. 10C). Exemplary CAR bindingschematics are depicted in FIGS. 10D-10E. The CAR may be expressed in alymphoid cell, e.g., a T cell, and an epitope to the CAR may beexpressed in a non-lymphoid cell, or in an in vitro matrix, such as abead, gel or column. See e.g., FIG. 10D. In some arrangements, the CARis capable of binding to an epitope of a “linker” protein or peptide,which is able to also serve as an epitope to a surface protein ofanother surface, e.g., a cell, bead, gel or column. See e.g., FIG. 10E.

Engineering Variance in Chimeric Antigen Receptor

Provided herein are methods for synthesis of variant nucleic acidlibraries, wherein each variant nucleic acid encodes for a sequence thatis varied in comparison to a reference domain within a CAR. For example,the varied sequence is a nucleic acid sequence that encodes for anantigen recognition domain, a hinge domain, a transmembrane domain, oran intracellular domain. In some instances, the intracellular domaincomprises a signaling domain. In some instances, the intracellulardomain further comprises a costimulatory domain. In some instances, thevariant nucleic acid libraries comprise variant sequences that encodefor all domains of a CAR. The resulting nucleic acid library may be anoligonucleotide library, gene fragment library, or gene library. In someinstances, the nucleic acid library is expressed in cells to generate avariant protein library.

Variant nucleic acid libraries generated by methods disclosed herein mayencode for an antigen recognition domain. An antigen recognition domainmay comprise a single chain variable fragment (scFv). In some instances,an antigen recognition domain comprises F(ab′)₂, Fab′, Fab, or Fv.Variant nucleic acid libraries that encode for antigen recognitiondomains may be designed and synthesized for a particular tumor antigen.Exemplary tumor antigens include, but are not limited to, α-Folatereceptor, BCMA, CAIX, CD123, CD138, CD171, CD19, CD20, CD22, CD30, CD33,CD44, CD44v7/8, CEA, cMET, DNAM-1, EDB-F, EGFR, EGFRvIII, EGP-2, EGP-40,EpCAM, FAP, Folate-binding Protein, GD2, GD3, glypican-3, h5T4, HER2,HER3, IL-13, IL-13R-a2, kappa light chain, KDR, Lewis Y, LMP-1, MAGE-A1,mesothelin, MUC-1, MUC-16, PSCA, PSMA, ROR1, TAG-72, VEGFR, and VEGFR2.In some instances, about 25, 50, 100, 250, 500, 1000, or more than 1000common coding gene sequences of an antigen recognition domain isselected for variation. Variation may include designing of at least 25,50, 100, 250, 500, 1000, 2000, 5000, 10000, 20000, 50000, 100000, ormore than 100000 variants for each of the common coding gene sequencesselected.

The hinge domain and transmembrane domain may connect the antigenrecognition domain to the intracellular domains and anchor the CAR inthe T cell membrane. In some instances, variant nucleic acid librariesare generated that encode for the hinge domain. Exemplary hinge domainsare immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgDor IgE), CH2CH3 region of immunoglobulin and optionally portions of CD3,and CD8α. In some cases, variant nucleic acid libraries are generatedthat encode for the transmembrane domain.

Variant nucleic acid libraries generated by methods disclosed herein maycomprise sequences that are varied to an intracellular domain. In someinstances, the intracellular domain comprises a costimulatory domain. Insome instances, variant nucleic acid libraries comprise sequences thatare varied to a reference costimulatory domain of a CAR. Exemplarycostimulatory domains include, but not limited to, CD8, CD27, CD28,4-1BB (CD137), ICOS, DAP10, OX40 (CD134) or fragments or combinationsthereof. In some instances, about 100, 250, 500, 1000, or more than 1000common coding gene sequences of a costimulatory domain is selected forvariation. Variation includes designing of at least about 500, 1000,2000, 5000, 10000, 20000, 50000, 100000, or more than 100000 variantsfor each of the common coding gene sequences selected. Exemplarycostimulatory domain sequences are shown in Table 1.

TABLE 1 Costimulatory Domains SEQ ID  Accession Name NO NumberNucleic Acid Sequence Human 24 AAL40933.1ATGAAGTCAGGCCTCTGGTATTTCTTTCTCTTCTG ICOSCTTGCGCATTAAAGTTTTAACAGGAGAAATCAATG GTTCTGCCAATTATGAGATGTTTATATTTCACAACGGAGGTGTACAAATTTTATGCAAATATCCTGACAT TGTCCAGCAATTTAAAATGCAGTTGCTGAAAGGGGGGCAAATACTCTGCGATCTCACTAAGACAAAAGGA AGTGGAAACACAGTGTCCATTAAGAGTCTGAAATTCTGCCATTCTCAGTTATCCAACAACAGTGTCTCTT TTTTTCTATACAACTTGGACCATTCTCATGCCAACTATTACTTCTGCAACCTATCAATTTTTGATCCTCC TCCTTTTAAAGTAACTCTTACAGGAGGATATTTGCATATTTATGAATCACAACTTTGTTGCCAGCTGAAG TTCTGGTTACCCATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGC TTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAA CACAGCCAAAAAATCTAGACTCACAGATGTGACCCTATAA Human 25 BAG60249.1. ATGGTATCACATCGGTATCCTCGAATTCAAAGTAT OX40CAAAGTACAATTTACCGAATATAAGAAGGAGAAA GGTTTCATCCTCACTTCCCAAAAGGAGGATGAAATCATGAAGGTGCAGAACAACTCAGTCATCATCAAC TGTGATGGGTTTTATCTCATCTCCCTGAAGGGCTACTTCTCCCAGGAAGTCAACATTAGCCTTCATTACC AGAAGGATGAGGAGCCCCTCTTCCAACTGAAGAAGGTCAGGTCTGTCAACTCCTTGATGGTGGCCTCTC TGACTTACAAAGACAAAGTCTACTTGAATGTGACCACTGACAATACCTCCCTGGATGACTTCCATGTGAA TGGCGGAGAACTGATTCTTATCCATCAAAATCCTGGTGAATTCTGTGTCCTTTGA Human 26 AAR05440.1ATGGGAAACAGCTGTTACAACATAGTAGCCACTCT 4-1BBGTTGCTGGTCCTCAACTTTGAGAGGACAAGATCAT (CD137)TGCAGGATCCTTGTAGTAACTGCCCAGCTGGTACA TTCTGTGATAATAACAGGAATCAGATTTGCAGTCCCTGTCCTCCAAATAGTTTCTCCAGCGCAGGTGGAC AAAGGACCTGTGACATATGCAGGCAGTGTAAAGGTGTTTTCAGGACCAGGAAGGAGTGTTCCTCCACCA GCAATGCAGAGTGTGACTGCACTCCAGGGTTTCACTGCCTGGGGGCAGGATGCAGCATGTGTGAACAGG ATTGTAAACAAGGTCAAGAACTGACAAAAAAAGGTTGTAAAGACTGTTGCTTTGGGACATTTAACGATC AGAAACGTGGCATCTGTCGACCCTGGACAAACTGTTCTTTGGATGGAAAGTCTGTGCTTGTGAATGGGA CGAAGGAGAGGGACGTGGTCTGTGGACCATCTCCAGCCGACCTCTCTCCGGGAGCATCCTCTGTGACCC CGCCTGCCCCTGCGAGAGAGCCAGGACACTCTCCGCAGATCATCTCCTTCTTTCTTGCGCTGACGTCGA CTGCGTTGCTCTTCCTGCTGTTCTTCCTCACGCTCCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAACT CCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGC CGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGTGA Human 27 AAR84239.1 ATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCT CD27GGGGACCCTGGTGGGGCTCTCAGCTACTCCAGCCC CCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCAGGGAAAGCTGTGCTGCCAGATGTGTGAGCCAGGA ACATTCCTCGTGAAGGACTGTGACCAGCATAGAAAGGCTGCTCAGTGTGATCCTTGCATACCGGGGGTC TCCTTCTCTCCTGACCACCACACCCGGCCCCACTGTGAGAGCTGTCGGCACTGTAACTCTGGTCTTCTCG TTCGCAACTGCACCATCACTGCCAATGCTGAGTGTGCCTGTCGCAATGGCTGGCAGTGCAGGGACAAGG AGTGCACCGAGTGTGATCCTCTTCCAAACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCACA CCCTCAGCCCACCCACTTACCTTATGTCAGTGAGATGCTGGAGGCCAGGACAGCTGGGCACATGCAGAC TCTGGCTGACTTCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCACCCCAAAGATCCCTGTGC AGCTCCGATTTTATTCGCATCCTTGTGATCTTCTCTGGAATGTTCCTTGTTTTCACCCTGGCCGGGGCCC TGTTCCTCCATCAACGAAGGAAATATAGATCAAACAAAGGAGAAAGTCCTGTGGAGCCTGCAGAGCCTT GTCGTTACAGCTGCCCCAGGGAGGAGGAGGGCAGCACCATCCCCATCCAGGAGGATTACCGAAAACCG GAGCCTGCCTGCTCCCCCTGA Human 28AAB04637.1 ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCT CD8GGCCTTGCTGCTCCACGCCGCCAGGCCGAGCCAGT alphaTCCGGGTGTCGCCGCTGGATCGGACCTGGAACCTG GGCGAGACAGTGGAGCTGAAGTGCCAGGTGCTGCTGTCCAACCCGACGTCGGGCTGCTCGTGGCTCTTC CAGCCGCGCGGCGCCGCCGCCAGTCCCACCTTCCTCCTATACCTCTCCCAAAACAAGCCCAAGGCGGCC GAGGGGCTGGACACCCAGCGGTTCTCGGGCAAGAGGTTGGGGGACACCTTCGTCCTCACCCTGAGCGAC TTCCGCCGAGAGAACGAGGGCTACTATTTCTGCTCGGCCCTGAGCAACTCCATCATGTACTTCAGCCACT TCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCA CCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACA CGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCT CCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACCGAAGACGTGTTTGCAAATGTCCCCGGCCT GTGGTCAAATCGGGAGACAAGCCCAGCCTTTCGGCGAGATACGTCTAA Human 29 AAD47911.1 ATGATCCATCTGGGTCACATCCTCTTCCTGCTTTTDAP10 GCTCCCAGTGGCTGCAGCTCAGACGACTCCAGGAGAGAGATCATCACTCCCTGCCTTTTACCCTGGCACT TCAGGCTCTTGTTCCGGATGTGGGTCCCTCTCTCTGCCGCTCCTGGCAGGCCTCGTGGCTGCTGATGCGG TGGCATCGCTGCTCATCGTGGGGGCGGTGTTCCTGTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGA TGGCAAAGTCTACATCAACATGCCAGGCAGGGGCTGA

Variant nucleic acid libraries as described herein may comprise variantsof the intracellular domain. In some instances, the intracellular domaincomprises a signaling domain. In some instances, variant nucleic acidlibraries generated by methods disclosed herein comprise sequences thatare varied when compared to the signaling domain. In some instances, thesignaling domain comprises an immunoreceptor tyrosine-based activationmotif (ITAM). In some instances, the signaling domain comprises a domainderived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3epsilon, CD5, CD22, CD79a, CD79b or CD66d. In some cases, the signalingdomain for T-cell activation comprises a domain derived from CD3ζ. Insome instances, about 100, 250, 500, 1000, or more than 1000 commoncoding gene sequences of a signaling domain for T cell activation isselected for variation. Variation includes designing of at least about500, 1000, 2000, 5000, 10000, 20000, 50000, 100000, or more than 100000variants for each of the common coding gene sequences selected.Exemplary signaling domain sequences are shown in Table 2.

TABLE 2 Signaling Domain Sequences SEQ ID Accession Name NO NumberNucleic Acid Sequence Human 30 AAI13831.1ATGGAACAGGGGAAGGGCCTGGCTGTCCTCATCCTGGCT CD3ATCATTCTTCTTCAAGGTACTTTGGCCCAGTCAATCAAA gammaGGAAACCACTTGGTTAAGGTGTATGACTATCAAGAAGATGGTTCGGTACTTCTGACTTGTGATGCAGAAGCCAAAAATATCACATGGTTTAAAGATGGGAAGATGATCGGCTTCCTAACTGAAGATAAAAAAAAATGGAATCTGGGAAGTAATGCCAAGGACCCTCGAGGGATGTATCAGTGTAAAGGATCACAGAACAAGTCAAAACCACTCCAAGTGTATTACAGAATGTGTCAGAACTGCATTGAACTAAATGCAGCCACCATATCTGGCTTTCTCTTTGCTGAAATCGTCAGCATTTTCGTCCTTGCTGTTGGGGTCTACTTCATTGCTGGACAGGATGGAGTTCGCCAGTCGAGAGCTTCAGACAAGCAGACTCTGTTGCCCAATGACCAGCTCTACCAGCCCCTCAAGGATCGAGAAGATGACCAGTACAGCCACCTTCAAGGAAACCAGTTGAGGAGGAAT TGA Human 31 EAW67364.1ATGCAGTCGGGCACTCACTGGAGAGTTCTGGGCCTCTGC CD3CTCTTATCAGTTGGCGTTTGGGGGCAAGATGGTAATGAA epsilonGAAATGGGTGGTATTACACAGACACCATATAAAGTCTCCATCTCTGGAACCACAGTAATATTGACATGCCCTCAGTATCCTGGATCTGAAATACTATGGCAACACAATGATAAAAACATAGGCGGTGATGAGGATGATAAAAACATAGGCAGTGATGAGGATCACCTGTCACTGAAGGAATTTTCAGAATTGGAGCAAAGTGGTTATTATGTCTGCTACCCCAGAGGAAGCAAACCAGAAGATGCGAACTTTTATCTCTACCTGAGGGCAAGAGTGTGTGAGAACTGCATGGAGATGGATGTGATGTCGGTGGCCACAATTGTCATAGTGGACATCTGCATCACTGGGGGCTTGCTGCTGCTGGTTTACTACTGGAGCAAGAATAGAAAGGCCAAGGCCAAGCCTGTGACACGAGGAGCGGGTGCTGGCGGCAGGCAAAGGGGACAAAACAAGGAGAGGCCACCACCTGTTCCCAACCCAGACTATGAGCCCATCCGGAAAGGCCAGCGGGACCTGTATTCTGGCCTGAATCAGAGACGCATCTGA Human 32 AAB20812.1ATGCCTGGGGGTCCAGGAGTCCTCCAAGCTCTGCCTGCC CD79ACCATCTTCCTCCTCTTCCTGCTGTCTGCTGTCTACCTG alphaGGCCCTGGGTGCCAGGCCCTGTGGATGCACAAGGTCCCAGCATCATTGATGGTGAGCCTGGGGGAAGACGCCCACTTCCAATGCCCGCACAATAGCAGCAACAACGCCAACGTCACCTGGTGGCGCGTCCTCCATGGCAACTACACGTGGCCCCCTGAGTTCTTGGGCCCGGGCGAGGACCCCAATGGTACGCTGATCATCCAGAATGTGAACAAGAGCCATGGGGGCATATACGTGTGCCGGGTCCAGGAGGGCAACGAGTCATACCAGCAGTCCTGCGGCACCTACCTCCGCGTGCGCCAGCCGCCCCCCAGGCCCTTCCTGGACATGGGGGAGGGCACCAAGAACCGAATCATCACAGCCGAGGGGATCATCCTCCTGTTCTGCGCGGTGGTGCCTGGGACGCTGCTGCTGTTCAGGAAACGATGGCAGAACGAGAAGCTCGGGTTGGATGCCGGGGATGAATATGAAGATGAAAACCTTTATGAAGGCCTGAACCTGGACGACTGCTCCATGTATGAGGACATCTCCCGGGGCCTCCAGGGCACCTACCAGGATGTGGGCAGCCTCAACATAGGAGATGTC CAGCTGGAGAAGCCGTGA Human 33NM_000626.3 AGGGGACAGGCTGCAGCCGGTGCAGTTACACGTTTTCCT CD79CCAAGGAGCCTCGGACGTTGTCACGGGTTTGGGGTCGGG betaGACAGAGCGGTGACCATGGCCAGGCTGGCGTTGTCTCCTGTGCCCAGCCACTGGATGGTGGCGTTGCTGCTGCTGCTCTCAGCTGAGCCAGTACCAGCAGCCAGATCGGAGGACCGGTACCGGAATCCCAAAGGTAGTGCTTGTTCGCGGATCTGGCAGAGCCCACGTTTCATAGCCAGGAAACGGGGCTTCACGGTGAAAATGCACTGCTACATGAACAGCGCCTCCGGCAATGTGAGCTGGCTCTGGAAGCAGGAGATGGACGAGAATCCCCAGCAGCTGAAGCTGGAAAAGGGCCGCATGGAAGAGTCCCAGAACGAATCTCTCGCCACCCTCACCATCCAAGGCATCCGGTTTGAGGACAATGGCATCTACTTCTGTCAGCAGAAGTGCAACAACACCTCGGAGGTCTACCAGGGCTGCGGCACAGAGCTGCGAGTCATGGGATTCAGCACCTTGGCACAGCTGAAGCAGAGGAACACGCTGAAGGATGGTATCATCATGATCCAGACGCTGCTGATCATCCTCTTCATCATCGTGCCTATCTTCCTGCTGCTGGACAAGGATGACAGCAAGGCTGGCATGGAGGAAGATCACACCTACGAGGGCCTGGACATTGACCAGACAGCCACCTATGAGGACATAGTGACGCTGCGGACAGGGGAAGTGAAGTGGTCTGTAGGTGAGCACCCAGGCCAGGAGTGAGAGCCAGGTCGCCCCATGACCTGGGTGCAGGCTCCCTGGCCTCAGTGACTGCTTCGGAGCTGCCTGGCTCATGGCCCAACCCCTTTCCTGGACCCCCCAGCTGGCCTCTGAAGCTGGCCCACCAGAGCTGCCATTTGTCTCCAGCCCCTGGTCCCCAGCTCTTGCCAAAGGGCCTGGAGTAGAAGGACAACAGGGCAGCAACTTGGAGGGAGTTCTCTGGGGATGGACGGGACCCAGCCTTCTGGGGGTGCTATGAGGTGATCCGTCCCCACACATGGGATGGGGGAGGCAGAGACTGGTCCAGAGCCCGCAAATGGACTCGGAGCCGAGGGCCTCCCAGCAGAGCTTGGGAAGGGCCATGGACCCAACTGGGCCCCAGAAGAGCCACAGGAACATCATTCCTCTCCCGCAACCACTCCCACCCCAGGGAGGCCCTGGCCTCCAGTGCCTTCCCCCGTGGAATAA ACGGTGTGTCCTGAGAAACCACACAAAAAAAAHuman 34 AAY57330.1 ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAG CD3GCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTG zetaCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTT CACATGCAGGCCCTGCCCCCTCGCTAA

Methods provided herein for the synthesis of a variant nucleic acidlibrary that is varied in comparison to a reference domain within a CARmay include: de novo synthesis of a variant library of primeroligonucleotides followed by PCR mutagenesis, or de novo synthesis ofmultiple fragments of the variant version for annealing and assemblyincluding polymerase chain assembly.

A nucleic acid library as described herein may comprise a plurality ofnucleic acids that are varied in comparison to a reference domain withina reference CAR, and variation is generated by de novo synthesis of avariant library of primer oligonucleotides followed by PCR mutagenesis.In some instances, oligonucleotides are synthesized on a surface,wherein each oligonucleotide encodes for a predetermined sequence. Insome instances, the predetermined sequence is a predetermined variant ofa reference nucleic acid sequence that encodes for an antigenrecognition domain, a hinge domain, a transmembrane domain, or anintracellular domain of a CAR. In some instances, oligonucleotideprimers are de novo synthesized for use in a series of PCR reactions togenerate a library of oligonucleotide variants of a reference nucleicacid sequence that encodes for an antigen recognition domain, a hingedomain, a transmembrane domain, or an intracellular domain of a CAR. Insome instances, the oligonucleotide primers are used for amplificationfrom a reference nucleic acid sequence to produce a library of variantnucleic acids encoding for an antigen recognition domain, a hingedomain, a transmembrane domain, or an intracellular domain of a CAR.

In some instances, a variant nucleic acid library varied in comparisonto a reference domain within a CAR is generated by de novo synthesis ofmultiple fragments of the variant version of the common sequence forannealing and assembly including polymerase chain assembly. In someinstances, a surface is used for de novo synthesis of multiple fragmentsof a nucleic acid sequence, wherein at least one of the fragments issynthesized in multiple versions. The nucleic acid sequence may be avariant of a reference nucleic acid sequence that encodes for an antigenrecognition domain, a hinge domain, a transmembrane domain, or anintracellular domain of a CAR. In some instances, the fragments aresubject to hybridization to generate a CAR variant library. In someinstances, the synthesized fragments are amplified and subject toligation or hybridization to generate a CAR variant library.

In some instances, a CAR variant library comprises nucleic acidsequences that encode for an antigen recognition domain, a hinge domain,a transmembrane domain, or an intracellular domain including astimulatory domain or a signaling domain of a CAR. In some instances, aCAR variant library comprises nucleic acid sequences that encodes for asingle domain of a CAR. In some instances, a CAR variant librarycomprises nucleic acid sequences that encodes for multiple domains of aCAR. For example, a CAR variant library comprises nucleic acid sequencesthat encode for all domains of a CAR.

Libraries comprising nucleic acids encoding for variant CARs asdescribed herein comprise various lengths of amino acids whentranslated. In some instances, the length of each of the amino acidfragments or average length of the amino acid synthesized may be atleast or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, ormore than 150 amino acids. In some instances, the length of the aminoacid is in a range of about 15 to 150, 20 to 145, 25 to 140, 30 to 135,35 to 130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to105, 70 to 100, or 75 to 95 amino acids. In some instances, the lengthof the amino acid is in a range of about 22 to about 75 amino acids.

A number of variant sequences for the at least one region of the CARchain for variation are de novo synthesized using methods as describedherein. In some instances, a number of variant sequences is de novosynthesized for an antigen recognition domain, a hinge domain, atransmembrane domain, or an intracellular domain of a CAR. In someinstances, a number of variant sequences is de novo synthesized for astimulatory domain or a signaling domain of a CAR. The number of variantsequences may be at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225,250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more than 500sequences. In some instances, the number of variant sequences is atleast or about 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000,90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000,800,000, 900,000, 1 million, or more than 1 million sequences. In someinstances, the number of variant sequences is in a range of about 10 to500, 25 to 475, 50 to 450, 75 to 425, 100 to 400, 125 to 375, 150 to350, 175 to 325, 200 to 300, 225 to 375, 250 to 350, or 275 to 325sequences.

Provided herein are variant nucleic acids encoding for variant CARs,wherein the variant CARs are antigen specific. In some instances, theantigen is involved in or associated with a disease, disorder, orcondition. For example, the antigen is associated with a proliferativedisease, a tumorous disease, an inflammatory disease, an immunologicaldisorder, an autoimmune disease, an infectious disease, a viral disease,an allergic reaction, a parasitic reaction, a graft-versus-host diseaseor a host-versus-graft disease. In some instances, the antigen is anantigen expressed on a tumor cell. In some instances, an antigen isassociated with a pathogen such as a virus or bacterium.

In some instances, the variant CARs recognize antigens that aretissue-restricted. For example, the variant CARs are restrictednon-vital cell lineages or tissues. In some instances, the variant CARsrecognize antigens from mutated gene products.

Provided herein are variant CAR libraries, wherein the variant CARsencode for variants in an antigen binding interface. In some instances,residues for variation are preselected or predicted residues thatcontact an antigen. In some instances, the antigen binding interface isthe antigen recognition domain. In some instances, residues forvariation are preselected or predicted residues located in the bindingpocket.

Variant CAR libraries as described herein comprise one or more mutationsin a library. In some instances, the CAR variant libraries are singlevariant libraries comprising variants at a single site across thelibrary. In some instances, the CAR variant libraries are multiplevariant libraries comprising variants at a number of sites. In someinstances, the number of sites is 2, 3, 4, 5, 6, 7, 8, 9, 10, or morethan 10 sites.

Provided here are libraries where one or more preselected codons in aCAR gene or gene fragment encode for variant amino acid residues togenerate variation in resulting variant CAR protein libraries. In someinstances, up to 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acidresidues are varied. In some instances, up to 30 amino acid residues arevaried. In some instances, up to 5 amino acid residues are varied. Insome instances, up to 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,or more than 1000 amino acid residues are varied. In some instances, atleast or about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acidresidues are varied. In some instances, at least or about 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, or more than 1000 amino acidresidues are varied. In some instances, all amino acid residues in apreselected region are varied. In some instances, variant CAR librariesare highly diverse. In some instances, the libraries comprise at leastor about 10{circumflex over ( )}6, 10{circumflex over ( )}8,10{circumflex over ( )}9, 10{circumflex over ( )}10, 10{circumflex over( )}11, 10{circumflex over ( )}12, or 10{circumflex over ( )}13variants. In some instances, the libraries comprise at least or about10{circumflex over ( )}9 variants.

Subsequent to synthesis of variant nucleic acid libraries encoding for areference domain within a CAR, variant nucleic acid libraries may beinserted into a vector sequence. In some instances, the vector sequenceis expressed (e.g., by electroporation, transfection, or transduction)in cells and functional consequences are determined. CAR domains thatmay be varied are an antigen recognition domain, a hinge domain, atransmembrane domain, or an intracellular domain. In some instances, theintracellular domain comprises a signaling domain or a costimulatorydomain. In some instances, the variant nucleic acid libraries comprisevariant sequences that encode for all domains of a CAR. Functionalconsequences that may be measured include binding affinity and bindingstrength. In some instances, variant nucleic acid libraries generated bymethods disclosed herein result in increased binding strength of anantigen recognition domain to a tumor antigen associated with aparticular cancer of interest. The cancer may be a solid cancer or ahematologic cancer. In some instances, the cancer is bladder cancer,lung cancer, brain cancer, melanoma, breast cancer, Non-Hodgkinlymphoma, cervical cancer, ovarian cancer, colorectal cancer, pancreaticcancer, esophageal cancer, prostate cancer, kidney cancer, skin cancer,leukemia, thyroid cancer, liver cancer, or uterine cancer. Exemplaryantigens for use in binding assays include, without limitation, thoseprovided in Table 3.

TABLE 3  Antigens Antigen SEQ peptide Protein ID name Sequence NODisease Target CD19 KVSAVTLAY 35 B-cell malignancy CD20 RPKSNIVLL 36B-cell malignancy CEA  IMIGVLVGV 37 Metastatic colorectal peptide cancerHER2 IISAVVGIL 38 Breast cancer, lung cancer, prostate cancer, gliomaNY-ESO- SLLMWITQV 39 Many cancers 1 including melanoma and sarcoma PSMAKYADKIYSI 40 Prostate cancer

Variant nucleic acid libraries generated using methods described hereinmay be screened to select a modified CAR domain sequence providing for aprotein complex with improved affinity (measure of the strength ofinteraction between an epitope and an antibody's antigen binding site)for a tumor antigen. Additional functional considerations, such asvariant gene expression, avidity, stability, and target cell (i.e.cancer cell) specificity may also be assessed. In some instances, theincreased specificity of a CAR provides for reduced cross-reactivity tonon-cancer associated antigens compared to a reference non-variant CAR.In some instances, the variant CAR libraries are screened forlocalization within a cell. In some instances, the variant CAR librariesare screened to identify properly localized variant CARs.

Increased specificity of a variant CAR to a tumor antigen may result indecreased toxicity. Toxicity in some instances that results includecytokine release syndrome (CRS), macrophage activation syndrome (MAS),on-target off-tumor toxicity, autoimmunity, host-graft toxicity,neurotoxicity, and tumor lysis syndrome. Variant nucleic acid librariesencoding for an antigen recognition domain may be inserted into a vectorsequence, expressed in cells, and screened for functional consequencessuch as decreased toxicity. For example, variant nucleic acid librariesare screened for increased cytokine (e.g., interleukin-6 (IL-6),interferon-γ, tumor necrosis factor, IL-2, IL-2—receptor-α, IL-8, andIL-10) release from cells or increased cytokine expression as a measureof CRS.

In some instances, variant nucleic acid libraries generated usingmethods described herein are screened to select a modified CAR domainsequence providing for improved T cell function or improved T cellactivation. In some instances, variant CARs are used to screen T cellpathways affecting cell growth, survival, cellular differentiation, celldeath, or intracellular signaling modulation. For example, theserine/threonine kinase Akt has been shown to improve T-cell functionand survival resulting in resistance to tumor inhibitory mechanisms. Insome cases, variant CARs are used to screen T cell pathways that improveresistance to tumor inhibitory mechanisms. In some instances, variantCARs generated using methods described herein result in enhanced immuneresponse towards a target cell such as a cancer cell. For example, avariant CAR may increase cytotoxic activity of a cytotoxic cell (e.g., aNatural Killer cell or cytotoxic T lymphocyte) towards a target cellsuch as a cancer cell. In some instances, variant CARs generated usingmethods described herein result in enhanced immune response towards atarget cell such as a cancer cell, thus resulting in increased death ofa cancer cell.

In some instances, variant CAR libraries that are expressed in cells areused to identify variant CARs with improved variant gene expression,avidity, stability, affinity, or specificity. For example, a firstvariant CAR library is generated that comprises improved specificity toa tumor antigen. In some instances, following identification of variantCARs with improved gene expression, avidity, stability, affinity, orspecificity, those variant CARs are further varied to produce a secondlibrary of variant CARs with a second improvement. For example, variantCARs with improved specificity are further varied to identify variantsfurther comprising improved stability. In some instances, the secondlibrary comprises variants in a same region as the first library. Insome instances, the second library comprises variants in a differentregion of the first library. For example, the first library may comprisevariants in an antigen recognition domain of the CAR and the secondlibrary may comprise variants in a hinge domain, a transmembrane domain,or an intracellular domain of the CAR. In some instances, a number ofvariant CAR libraries are generated. In some instances, the number ofvariant CAR libraries is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, ormore than 12 variant libraries. In some instances, the variant librariescomprise at least or about 10¹, 10², 10³, 10⁴, 10⁵, or 10⁶ variants. Insome instances, each of the variant libraries independently hasimprovements in gene expression, avidity, stability, affinity, orspecificity.

Variant CAR libraries may be engineered into cells and introduced into asubject. In some instances, the cells are autologous, meaning derivedfrom a subject's own cells. Alternately, cells expressing variant CARsare allogeneic, meaning derived from another subject with a similartissue type. In some instances, cells are tailored to the subject. Insome instances, cells are compatible with local tissue.

In some instances, variant nucleic acid libraries as described hereincomprise about 50-100000, 100-75000, 250-50000, 500-25000, 1000-15000,2000-10000, or 4000-8000 sequences. In some instances, variant nucleicacid libraries comprise 500 sequences. In some cases, variant nucleicacid libraries comprise 5000 sequences. In some instances, variantnucleic acid libraries comprise 15000 sequences. In some cases, variantnucleic acid libraries comprise at least 50, 100, 150, 500, 1000, 2000,5000, 10000, 20000, 50000, 100000, 200000, 400000, 800000, 1000000, ormore than 1000000 sequences. Variant nucleic acid libraries may compriseat most 50, 100, 500, 1000, 2000, 5000, 10000, 20000, 50000, 100000,200000, 400000, 800000, or1000000 sequences. The total variant librarymay result in 10{circumflex over ( )}6, 10{circumflex over ( )}8,10{circumflex over ( )}10, 10{circumflex over ( )}11, 10{circumflex over( )}12, 10{circumflex over ( )}13 or more different nucleic acids.

In some cases, the length of each of the oligonucleotide fragments oraverage length of the oligonucleotides synthesized may be at least orabout at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 300,400, 500, 2000 nucleotides, or more. The length of each of theoligonucleotide fragments or average length of the oligonucleotidessynthesized may be at most or about at most 2000, 500, 400, 300, 200,150, 100, 50, 45, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10nucleotides, or less. The length of each of the oligonucleotidefragments or average length of the oligonucleotides synthesized may fallfrom 10-2000, 10-500, 9-400, 11-300, 12-200, 13-150, 14-100, 15-50,16-45, 17-40, 18-35, 19-25.

Variant Library Synthesis

Methods described herein provide for synthesis of a library ofoligonucleotides each encoding for a predetermined variant of at leastone predetermined reference nucleic acid sequence. In some instances,the oligonucleotides synthesized herein are isolated, purified, orisolated and purified. In some cases, the predetermined referencesequence is a nucleic acid sequence encoding for a protein, and thevariant library comprises sequences encoding for variation of at least asingle codon such that a plurality of different variants of a singleresidue in the subsequent protein encoded by the synthesized nucleicacid are generated by standard translation processes. The synthesizedspecific alterations in the nucleic acid sequence may be introduced byincorporating nucleotide changes into overlapping or blunt endedoligonucleotide primers. Alternatively, a population of oligonucleotidesmay collectively encode for a long nucleic acid (e.g., a gene) andvariants thereof. In this arrangement, the population ofoligonucleotides may be hybridized and subject to standard molecularbiology techniques to form the long nucleic acid (e.g., a gene) andvariants thereof. When the long nucleic acid (e.g., a gene) and variantsthereof are expressed in cells, a variant protein library is generated.Similarly, provided here are methods for synthesis of variant librariesencoding for RNA sequences (e.g., miRNA, shRNA, and mRNA) or DNAsequences (e.g., enhancer, promoter, UTR, and terminator regions). Insome instances, the sequences are exon sequences or coding sequences. Insome instances, the sequences do not comprise intron sequences. Alsoprovided here are downstream applications for variants selected out ofthe libraries synthesized using methods described here. Downstreamapplications include identification of variant nucleic acid or proteinsequences with enhanced biologically relevant functions, e.g.,biochemical affinity, enzymatic activity, changes in cellular activity,and for the treatment or prevention of a disease state.

Synthesis Followed by PCR Mutagenesis

A first process for synthesis of a variant library of oligonucleotidesis for PCR mutagenesis (saturating or non-saturating) methods. In thisworkflow, a plurality of oligonucleotides is synthesized, wherein eacholigonucleotide encodes for a predetermined sequence which is apredetermined variant of a reference nucleic acid sequence. Referring tothe figures, an exemplary workflow in depicted in FIGS. 1A-1D, whereinoligonucleotides are generated on a surface. FIG. 1A depicts anexpansion view of a single cluster of a surface with 121 loci. Eacholigonucleotide depicted in FIG. 1B is a primer that may be used foramplification from a reference nucleic acid sequence to produce alibrary of variant long nucleic acids, FIG. 1C. The library of variantlong nucleic acids is then, optionally, subject to transcription and ortranslation to generate a variant RNA or protein library, FIG. 1D. Inthis exemplary illustration, a device having a substantially planarsurface used for de novo synthesis of oligonucleotides is depicted, FIG.1A. In some instances, the device comprises a cluster of loci, whereineach locus is a site for oligonucleotide extension. In some instances, asingle cluster comprises all the oligonucleotide variants needed togenerate a desired variant sequence library. In an alternativearrangement, a plate comprises a field of loci which are not segregatedinto clusters.

In some instances, oligonucleotides synthesized within a cluster (e.g.,as seen in FIG. 1A) are amplified by PCR. Such an arrangement mayprovide for improved oligonucleotide representation compared toamplification of non-identical oligonucleotides across an entire platewithout a clustered arrangement. In some instances, amplification ofoligonucleotides synthesized on surfaces of loci within a clusterovercomes negative effects on representation due to repeated synthesisof large oligonucleotide populations having oligonucleotides with heavyGC content. In some instances, a cluster described herein, comprisesabout 50-1000, 75-900, 100-800, 125-700, 150-600, 200-500, 50-500 or300-400 discrete loci. In some instances, a loci is a spot, well,microwell, channel, or post. In some instances, each cluster has atleast 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, or more redundancy ofseparate features supporting extension of oligonucleotides having anidentical sequence. In some instances, 1× redundancy means not havingoligonucleotides with the same sequence.

A de novo synthesized oligonucleotide library described herein maycomprise a plurality of oligonucleotides, each with at least one variantsequence at a first position, position “x”, and each variantoligonucleotide is used as a primer in a first round of PCR to generatea first extension product. In this example, position “x” in a firstoligonucleotide 220 encodes for a variant codon sequence, i.e., one of19 possible variants from a reference sequence. See FIG. 2A. A secondoligonucleotide 225 comprising a sequence overlapping that of the firstoligonucleotide is also used as a primer in a separate round of PCR togenerate a second extension product. In addition, outer primers 215, 230may be used for amplification of a fragment from a long nucleic acidsequence. The resultant amplification products are fragments of the longnucleic acid sequence 235, 240. See FIG. 2B. The fragments of the longnucleic acid sequence 235, 240 are then hybridized, and subject to anextension reaction to form a variant of the long nucleic acid 245. SeeFIG. 2C. The overlapping ends of the first and second extension productsmay serve as a primer of a second round of PCR, thereby generating athird extension product (FIG. 2D) that contains the variant. To increasethe yield, the variant of the long nucleic acid is amplified in areaction including a DNA polymerase, amplification reagents, and theouter primers 215, 230. In some instances, the second oligonucleotidecomprises a sequence adjacent to, but not including, the variant site.In an alternative arrangement, a first oligonucleotide is generated thathas a region that overlaps with a second oligonucleotide. In thisscenario, the first oligonucleotide is synthesized with variation at asingle codon for up to 19 variants. The second oligonucleotide does notcomprise a variant sequence. Optionally, a first population comprisesthe first oligonucleotide variants and additional oligonucleotidesencoding for variants at a different codon site. Alternatively, thefirst oligonucleotide and the second oligonucleotide may be designed forblunt end ligation.

An alternative mutagenesis PCR method is depicted in FIGS. 3A-3F. Insuch a process, a template nucleic acid molecule 300 comprising a firstand second strand 305, 310 is amplified in a PCR reaction containing afirst primer 315 and a second primer 320 (FIG. 3A). The amplificationreaction includes uracil as a nucleotide reagent. A uracil-labeledextension product 325 (FIG. 3B) is generated, optionally purified, andserves as a template for a subsequent PCR reaction using a firstoligonucleotide 335 and a plurality of second oligonucleotides 330 togenerate first extension products 340 and 345 (FIGS. 3C-3D). In thisprocess, a plurality of second oligonucleotide 330 comprisesoligonucleotides encoding for variant sequences (denoted as X, Y, and Z,in FIG. 3C). The uracil-labeled template nucleic acid is digested by auracil-specific excision reagent, e.g., USER digest availablecommercially from New England Biolabs. Variant 335 and different codons330 with variants X, Y, and Z are added and a limited PCR step isperformed to generate FIG. 3D. After the uracil-containing template isdigested, the overlapping ends of the extension products serve to primea PCR reaction with the first extension products 340 and 345 acting asprimers in combination with a first outer primer 350 and a second outerprimer 355, thereby generating a library of nucleic acid molecules 360containing a plurality of variants X, Y, and Z at the variant site FIG.3F.

De Novo Synthesis of a Population With Variant and Non-Variant Portionsof a Long Nucleic Acid

In a second process for synthesis of a variant library, a surface isused for de novo synthesis of multiple fragments of a long nucleic acid,wherein at least one of the fragments is synthesized in multipleversions, each version being of a different variant sequence. In thisarrangement, all of the fragments needed to assemble a library ofvariant long range nucleic acids are de novo synthesized. Thesynthesized fragments may have overlapping sequence such that, followingsynthesis, the fragment library is subject to hybridization. Followinghybridization, an extension reaction may be performed to fill in anycomplementary gaps.

Alternatively, the synthesized fragments may be amplified with primersand then subject to either blunt end ligation or overlappinghybridization. In some instances, the device comprises a cluster ofloci, wherein each locus is a site for oligonucleotide extension. Insome instances, a single cluster comprises all the oligonucleotidevariants and other fragment sequences of a predetermined long nucleicacid to generate a desired variant nucleic acid sequence library. Thecluster may comprise about 50 to 500 loci. In some arrangements, acluster comprises greater than 500 loci.

Each individual oligonucleotide in the first oligonucleotide populationmay be generated on a separate, individually addressable locus of acluster. One oligonucleotide variant may be represented by a pluralityof individually addressable loci. Each variant in the firstoligonucleotide population may be represented 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more times. In some instances, each variant in the firstoligonucleotide population is represented at 3 or less loci. In someinstances, each variant in the first oligonucleotide population isrepresented at two loci. In some instances, each variant in the firstoligonucleotide population is represented at only a single locus.

Methods are provided herein to generate nucleic acid libraries withreduced redundancy. In some instances, variant oligonucleotides may begenerated without the need to synthesize the variant oligonucleotidemore than 1 time to obtain the desired variant oligonucleotide. In someinstances, the present disclosure provides methods to generate variantoligonucleotides without the need to synthesize the variantoligonucleotide more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more timesto generate the desired variant oligonucleotide.

Variant oligonucleotides may be generated without the need to synthesizethe variant oligonucleotide at more than 1 discrete site to obtain thedesired variant oligonucleotide. The present disclosure provides methodsto generate variant oligonucleotides without the need to synthesize thevariant oligonucleotide at more than 1 site, 2 sites, 3 sites, 4 sites,5 sites, 6 sites, 7 sites, 8 sites, 9 sites, or 10 sites. In someinstances, an oligonucleotide is synthesized in at most 6, 5, 4, 3, 2,or 1 discrete sites. The same oligonucleotide may be synthesized in 1,2, or 3 discrete loci on a surface.

In some instances, the amount of loci representing a single variantoligonucleotide is a function of the amount of nucleic acid materialrequired for downstream processing, e.g., an amplification reaction orcellular assay. In some instances, the amount of loci representing asingle variant oligonucleotide is a function of the available loci in asingle cluster.

Provided herein are methods for generation of a library ofoligonucleotides comprising variant oligonucleotides differing at aplurality of sites in a reference nucleic acid. In such cases, eachvariant library is generated on an individually addressable locus withina cluster of loci. It will be understood that the number of variantsites represented by the oligonucleotide library will be determined bythe number of individually addressable loci in the cluster and thenumber of desired variants at each site. In some instances, each clustercomprises about 50 to 500 loci. In some instances, each clustercomprises 100 to 150 loci.

In an exemplary arrangement, 19 variants are represented at a variantsite corresponding to codons encoding for each of the 19 possiblevariant amino acids. In another exemplary case, 61 variants arerepresented at a variant site corresponding to triplets encoding foreach of the 19 possible variant amino acids. In a non-limiting example,a cluster comprises 121 individually addressable loci. In this example,an oligonucleotide population comprises 6 replicates each of asingle-site variant (6 replicates×1 variant site×19 variants=114 loci),3 replicates each of a double-site variant (3 replicates×2 variantsites×19 variants=114 loci), or 2 replicates each of a triple-sitevariant (2 replicates×3 variant sites×19 variants=114 loci). In someinstances, an oligonucleotide population comprises variants at four,five, six or more than six variant sites.

Provided herein are methods and compositions for production of synthetic(i.e. de novo synthesized or chemically synthesized) oligonucleotides.Libraries of synthesized oligonucleotides described herein may comprisea plurality of oligonucleotides collectively encoding for one or moregenes or gene fragments. In some instances, the oligonucleotide librarycomprises coding or non-coding sequences. In some instances, theoligonucleotide library encodes for a plurality of cDNA sequences. Insome instances, the oligonucleotide library comprises one or moreoligonucleotides, each of the one or more oligonucleotides encodingsequences for multiple exons. Each oligonucleotide within a librarydescribed herein may encode a different sequence, i.e., non-identicalsequence. In some instances, each oligonucleotide within a librarydescribed herein comprises at least one portion that is complementary toa sequence of another oligonucleotide within the library.Oligonucleotide sequences described herein may, unless stated otherwise,comprise DNA or RNA.

Provided herein are methods and compositions for production of synthetic(i.e. de novo synthesized) genes. Libraries comprising synthetic genesmay be constructed by a variety of methods described in further detailelsewhere herein, such as PCA, non-PCA gene assembly methods orhierarchical gene assembly, combining (“stitching”) two or moredouble-stranded oligonucleotides to produce larger DNA units (i.e., achassis). Libraries of large constructs may involve oligonucleotidesthat are at least 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40,50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500 kb longor longer. The large constructs may be bound by an independentlyselected upper limit of about 5000, 10000, 20000 or 50000 base pairs.The synthesis of any number of polypeptide-segment encoding nucleotidesequences may include sequences encoding non-ribosomal peptides (NRPs),sequences encoding non-ribosomal peptide-synthetase (NRPS) modules andsynthetic variants, polypeptide segments of other modular proteins, suchas antibodies, polypeptide segments from other protein families,including non-coding DNA or RNA, such as regulatory sequences e.g.promoters, transcription factors, enhancers, siRNA, shRNA, RNAi, miRNA,small nucleolar RNA derived from microRNA, or any functional orstructural DNA or RNA unit of interest. The following are non-limitingexamples of oligonucleotides: coding or non-coding regions of a gene orgene fragment, intergenic DNA, loci (locus) defined from linkageanalysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomalRNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA(miRNA), small nucleolar RNA, ribozymes, cDNA, which is a DNArepresentation of mRNA, usually obtained by reverse transcription ofmessenger RNA (mRNA) or by amplification; DNA molecules producedsynthetically or by amplification, genomic DNA, recombinantoligonucleotides, branched oligonucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probes,and primers. In the context of cDNA, the term gene or gene fragmentrefers to a DNA nucleic acid sequence comprising at least one regionencoding for exon sequences without an intervening intron sequence.

In various embodiments, methods and compositions described herein relateto a library of genes. The gene library may comprise a plurality ofsubsegments. In one or more subsegments, the genes of the library may becovalently linked together. In one or more subsegments, the genes of thelibrary may encode for components of a first metabolic pathway with oneor more metabolic end products. In one or more subsegments, genes of thelibrary may be selected based on the manufacturing process of one ormore targeted metabolic end products. The one or more metabolic endproducts may comprise a biofuel. In one or more subsegments, the genesof the library may encode for components of a second metabolic pathwaywith one or more metabolic end products. The one or more end products ofthe first and second metabolic pathways may comprise one or more sharedend products. In some cases, the first metabolic pathway comprises anend product that is manipulated in the second metabolic pathway.

Codon Variation

Variant oligonucleotide libraries described herein may comprise aplurality of oligonucleotides, wherein each oligonucleotide encodes fora variant codon sequence compared to a reference nucleic acid sequence.In some instances, each oligonucleotide of a first oligonucleotidepopulation contains a variant at a single variant site. In someinstances, the first oligonucleotide population contains a plurality ofvariants at a single variant site such that the first oligonucleotidepopulation contains more than one variant at the same variant site. Thefirst oligonucleotide population may comprise oligonucleotidescollectively encoding multiple codon variants at the same variant site.The first oligonucleotide population may comprise oligonucleotidescollectively encoding up to 19 or more codons at the same position. Thefirst oligonucleotide population may comprise oligonucleotidescollectively encoding up to 60 variant triplets at the same position, orthe first oligonucleotide population may comprise oligonucleotidescollectively encoding up to 61 different triplets of codons at the sameposition. Each variant may encode for a codon that results in adifferent amino acid during translation. Table 4 provides a listing ofeach codon possible (and the representative amino acid) for a variantsite.

TABLE 4 List of codons and amino acids One Three letter letter AminoAcids code code Codons Alanine A Ala GCA GCC GCG GCT Cysteine C Cys TGCTGT Aspartic acid D Asp GAC GAT Glutamic acid E Glu GAA GAGPhenylalanine F Phe TTC TTT Glycine G Gly GGA GGC GGG GGT Histidine HHis CAC CAT Isoleucine I Iso ATA ATC ATT Lysine K Lys AAA AAG Leucine LLeu TTA TTG CTA CTC CTG CTT Methionine M Met ATG Asparagine N Asn AACAAT Proline P Pro CCA CCC CCG CCT Glutamine Q Gln CAA CAG Arginine R ArgAGA AGG CGA CGC CGG CGT Serine S Ser AGC AGT TCA TCC TCG TCT Threonine TThr ACA ACC ACG ACT Valine V Val GTA GTC GTG GTT Tryptophan W Trp TGGTyrosine Y Tyr TAC TAT

Provided herein are nucleic acid libraries that may comprise a pluralityof nucleic acids, wherein each nucleic acid encodes for a variant codonsequence compared to a reference nucleic acid sequence. In someinstances, each nucleic acid of a first nucleic acid population containsa variant at a single variant site. In some instances, the first nucleicacid population contains a plurality of variants at a single variantsite such that the first nucleic acid population contains more than onevariant at the same variant site. The first nucleic acid population maycomprise nucleic acids collectively encoding multiple codon variants atthe same variant site. The first oligonucleotide population may comprisenucleic acids collectively encoding up to 19 or more codons at the sameposition. The first nucleic acid population may comprise nucleic acidscollectively encoding up to 60 variant triplets at the same position, orthe first nucleic acid population may comprise oligonucleotidescollectively encoding up to 61 different triplets of codons at the sameposition. Each variant may encode for a codon that results in adifferent amino acid during translation.

An oligonucleotide population may comprise varied oligonucleotidescollectively encoding up to 20 codon variations at multiple positions.In such cases, each oligonucleotide in the population comprisesvariation for codons at more than one position in the sameoligonucleotide. In some instances, each oligonucleotide in thepopulation comprises variation for codons at 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more codons in a singleoligonucleotide. In some instances, each variant long nucleic acidcomprises variation for codons at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30or more codons in a single long nucleic acid. In some instances, thevariant oligonucleotide population comprises variation for codons at 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30 or more codons in a singleoligonucleotide. In some instances, the variant oligonucleotidepopulation comprises variation for codons in at least about 10, 20, 30,40, 50, 60, 70, 80, 90, 100 or more codons in a single long nucleicacid.

Provided herein are processes where a second oligonucleotide populationis generated on a second cluster containing a plurality of individuallyaddressable loci. The second oligonucleotide population may comprise aplurality of second oligonucleotides that are constant for each codonposition (i.e., encode the same amino acid at each position). The secondoligonucleotide may overlap with at least a portion of the firstoligonucleotides. In some instances, the second oligonucleotides do notcontain the variant site represented on the first oligonucleotides.Alternatively, the second oligonucleotide population may comprise aplurality of second oligonucleotides that contain at least one variantfor one or more codon positions.

Provided herein are methods for synthesizing a library ofoligonucleotides where a single population of oligonucleotides isgenerated comprising variants at multiple codon positions. A firstoligonucleotide population may be generated on a first clustercontaining a plurality of individually addressable loci. In such cases,the first oligonucleotide population comprises variants at differentcodon positions. In some instances, the different sites are consecutive(i.e., encoding consecutive amino acids). For example, the firstoligonucleotide population comprises variants in two consecutive codonpositions, encoding up to 19 variants at a position. In some instances,the first oligonucleotide population comprises variants in twoconsecutive codon positions, encoding from about 1 to about 19 variantsat a position. In some instances, about 38 oligonucleotides aresynthesized. In some instances, the oligonucleotides comprising variantsin two consecutive codon positions comprise a range of about 36 to about66 bases in length. A first oligonucleotide population may comprisevaried oligonucleotides collectively encoding up to 19 codon variants atthe same, or additional variant site. A first oligonucleotide populationmay include a plurality of first oligonucleotides that contains up to 19variants at position x, up to 19 variants at position y, and up to 19variants at position z. In such an arrangement, each variant encodes adifferent amino acid such that up to 19 amino acid variants are encodedat each of the different variant sites. In an additional instance, asecond oligonucleotide population is generated on a second clustercontaining a plurality of individually addressable loci. The secondoligonucleotide population may comprise a plurality of secondoligonucleotides that are constant for each codon position (i.e., encodethe same amino acid at each position). The second oligonucleotides mayoverlap with at least a portion of the first oligonucleotides. Thesecond oligonucleotides may not contain the variant site represented onthe first oligonucleotides.

Variant nucleic acid libraries generated by processes described hereinprovide for the generation of variant protein libraries. In a firstexemplary arrangement, a template oligonucleotide encodes for a sequencethat, when transcribed and translated, results in a reference amino acidsequence (FIG. 4A) having a number of codon positions, indicated by asingle circle. Oligonucleotide variants of the template may be generatedusing methods described herein. In some instances, a single variant ispresent in the oligonucleotide, resulting in a single amino acidsequence (FIG. 4B). In some instances, more than one variant is presentin the oligonucleotide, wherein the variants are separated by one ormore codons, resulting in a protein with spacing between variantresidues (FIG. 4C). In some instances, more than one variant is presentin the oligonucleotide, wherein the variants are sequential and adjacentor consecutive to one another, resulting in spaced variant stretches ofresidues (FIG. 4D). In some instances, two stretches of variants arepresent in the oligonucleotide, wherein each stretch of variantscomprises sequential and adjacent or consecutive variants (FIG. 4E).

Provided herein are methods to generate a library of oligonucleotidevariants, wherein each variant comprises a single position codonvariant. In one instance, a template oligonucleotide has a number ofcodon positions wherein exemplary amino acid residues are indicated bycircles with their respective one letter code protein codon, FIG. 5A.FIG. 5B depicts a library of amino acid variants encoded by a library ofvariant nucleic acids, wherein each variant comprises a single positionvariant, indicated by an “X”, located at a different single site. Afirst position variant has any codon to replace alanine, a secondvariant with any codon encoded by the library of variant nucleic acidsto replace tryptophan, a third variant with any codon to replaceisoleucine, a fourth variant with any codon to replace lysine, a fifthvariant with any codon to replace arginine, a sixth variant with anycodon to replace glutamic acid, and a seventh variant with any codon toreplace glutamine. When all or less than all codon variants are encodedby the variant nucleic acid library, a resulting correspondingpopulation of amino acid sequence variants is generated followingprotein expression (i.e., standard cellular events of DNA transcriptionfollowed by translation and processing events).

In some arrangements, a library is generated with multiple sites ofsingle position variants. As depicted in FIG. 6A, a wild-type templateis provided. FIG. 6B depicts the resultant amino acid sequence with twosites of single position codon variants, wherein each codon variantencoding for a different amino acid is indicated by differentlypatterned circles.

Provided herein are methods to generate a library having a stretch ofmultiple site, single position variants. Each stretch of oligonucleotideor nucleic acid may have 1, 2, 3, 4, 5, or more variants. Each stretchof oligonucleotide or nucleic acid may have at least 1 variant. Eachstretch of oligonucleotide or nucleic acid may have at least 2 variants.Each stretch of oligonucleotide or nucleic acid may have at least 3variants. For example, a stretch of 5 oligonucleotides or nucleic acidsmay have 1 variant. A stretch of 5 oligonucleotides or nucleic acids mayhave 2 variants. A stretch of 5 oligonucleotides or nucleic acids mayhave 3 variants. A stretch of 5 oligonucleotides or nucleic acids mayhave 4 variants. For example, a stretch of 4 oligonucleotides or nucleicacids may have 1 variant. A stretch of 4 oligonucleotides or nucleicacids may have 2 variants. A stretch of 4 oligonucleotides or nucleicacids may have 3 variants. A stretch of 4 oligonucleotides or nucleicacids may have 4 variants.

In some instances, single position variants may all encode for the sameamino acid, e.g. a histidine. As depicted in FIG. 7A, a reference aminoacid sequence is provided. In this arrangement, a stretch of anoligonucleotide encodes for multiple sites of single position variantsand, when expressed, results in an amino acid sequence having all singleposition variants encoding for a histidine, FIG. 7B. In someembodiments, a variant library synthesized by methods described hereindoes not encode for more than 4 histidine residues in a resultant aminoacid sequence.

In some instances, a variant library of nucleic acids generated bymethods described herein provides for expression of amino acid sequenceshaving separate stretches of variation. A template amino acid sequenceis depicted in FIG. 8A. A stretch of oligonucleotides may have only 1variant codon in two stretches and, when expressed, result in an aminoacid sequence depicted in FIG. 8B. Variants are depicted in FIG. 8B bythe differently patterned circles to indicate variation in amino acidsat different positions in a single stretch.

Provided herein are methods and devices to synthesize oligonucleotide ornucleic acid libraries with 1, 2, 3, or more codon variants, wherein thevariant for each site is selectively controlled. The ratio of two aminoacids for a single site variant may be about 1:100, 1:50, 1:10, 1:5,1:3, 1:2, 1:1. The ratio of three amino acids for a single site variantmay be about 1:1:100, 1:1:50, 1:1:20, 1:1:10, 1:1:5, 1:1:3, 1:1:2,1:1:1, 1:10:10, 1:5:5, 1:3:3, or 1:2:2. FIG. 9A depicts a wild-typereference amino acid sequence encoded by a wild-type nucleic acidsequence. FIG. 9B depicts a library of amino acid variants, wherein eachvariant comprising a stretch of sequence (indicated by the patternedcircles), wherein each position may have a certain ratio of amino acidsin the resultant variant protein library. The resultant variant proteinlibrary is encoded by a variant nucleic acid library generated bymethods described herein. In this illustration, 5 positions are varied:the first position 900 has a 50/50 K/R ratio; the second position 910has a 50/25/25 V/L/S ratio, the third position 920 has a 50/25/25 Y/R/Dratio, the fourth position 930 has an equal ratio for all 20 aminoacids, and the fifth position 940 has a 75/25 ratio for G/P. The ratiosdescribed herein are exemplary only.

Variation in Expression Cassettes

In some instances, a synthesized variant library is generated whichencodes for a portion of an expression construct. Exemplary portions ofan expression construct include the promoter, open reading frame, andtermination region. In some instances, the expression construct encodesfor one, two, three or more expression cassettes. An oligonucleotidelibrary may be generated, encoding for codon variation at a single siteor multiple sites in separate regions that make up portions of anexpression construct cassette, as depicted in FIG. 11. To generate a twoconstruct expressing cassette, variant oligonucleotides were synthesizedencoding at least a portion of a variant sequence of a first promoter1110, first open reading frame 1120, first terminator 1130, secondpromoter 1140, second open reading frame 1150, or second terminatorsequence 1160. After rounds of amplification, as described in previousexamples, a library of 1,024 expression constructs was generated. FIG.11 provides but one example arrangement. In some instances, additionalregulator sequences, such as untranslated regulatory region (UTR) or anenhancer region, are also included in an expression cassette referred toherein. An expression cassette may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more components for which variant sequences are generated bymethods described herein. In some instances, the expression constructcomprises more than one gene in a multicistronic vector. In one example,the synthesized DNA nucleic acids are inserted into viral vectors (e.g.,a lentivirus) and then packaged for transduction into cells, ornon-viral vectors for transfer into cells, followed by screening andanalysis.

Expression vectors for inserting nucleic acids disclosed herein comprisemammalian cells, e.g., human, non-human primate, pig, rabbit and mouse.Exemplary expression vectors include, without limitation, mammalianexpression vectors: pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF-CMV-NEO-COOH-3XFLAG,pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV-FLAG(R)-6His (“6His”disclosed as SEQ ID NO: 41), pCEP4 pDEST27, pSF-CMV-Ub-KrYFP,pSF-CMV-FMDV-daGFP, pEF1a-mCherry-N1 Vector, pEF1a-tdTomato Vector,pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro, pMCP-tag(m), andpSF-CMV-PURO-NH2-CMYC. Nucleic acids synthesized by methods describedherein may be transferred into cells by various methods known in theart, including, without limitation, transfection, transduction, andelectroporation. Exemplary cellular functions tested include, withoutlimitation, changes in cellular proliferation, death,migration/adhesion, metabolic, and cell-signaling activity.

Highly Parallel Nucleic Acid Synthesis

Provided herein is a platform approach utilizing miniaturization,parallelization, and vertical integration of the end-to-end process fromoligonucleotide synthesis to gene assembly within nanowells on siliconto create a revolutionary synthesis platform. Devices described hereinprovide, with the same footprint as a 96-well plate, a silicon synthesisplatform capable of increasing throughput by a factor of up to 1,000 ormore compared to traditional synthesis methods, with production of up toapproximately 1,000,000 or more oligonucleotides, or 10,000 or moregenes in a single highly-parallelized run.

With the advent of next-generation sequencing, high resolution genomicdata has become an important factor for studies that delve into thebiological roles of various genes in both normal biology and diseasepathogenesis. At the core of this research is the central dogma ofmolecular biology and the concept of “residue-by-residue transfer ofsequential information.” Genomic information encoded in the DNA istranscribed into a message that is then translated into the protein thatis the active product within a given biological pathway.

Another exciting area of study is on the discovery, development andmanufacturing of therapeutic molecules focused on a highly-specificcellular target. High diversity DNA sequence libraries are at the coreof development pipelines for targeted therapeutics. Gene mutants areused to express proteins in a design, build, and test proteinengineering cycle that ideally culminates in an optimized gene for highexpression of a protein with high affinity for its therapeutic target.As an example, consider the binding pocket of a receptor. The ability totest all sequence permutations of all residues within the binding pocketsimultaneously will allow for a thorough exploration, increasing chancesof success. Saturation mutagenesis, in which a researcher attempts togenerate all possible mutations at a specific site within the receptor,represents one approach to this development challenge. Though costly andtime and labor-intensive, it enables each variant to be introduced intoeach position. In contrast, combinatorial mutagenesis, where a fewselected positions or short stretch of DNA may be modified extensively,generates an incomplete repertoire of variants with biasedrepresentation.

To accelerate the drug development pipeline, a library with the desiredvariants available at the intended frequency in the right positionavailable for testing—in other words, a precision library, enablesreduced costs as well as turnaround time for screening. Provided hereinare methods for synthesizing nucleic acid synthetic variant librarieswhich provide for precise introduction of each intended variant at thedesired frequency. To the end user, this translates to the ability tonot only thoroughly sample sequence space but also be able to querythese hypotheses in an efficient manner, reducing cost and screeningtime. Genome-wide editing may elucidate important pathways, librarieswhere each variant and sequence permutation may be tested for optimalfunctionality, and thousands of genes may be used to reconstruct entirepathways and genomes to re-engineer biological systems for drugdiscovery.

In a first example, a drug itself may be optimized using methodsdescribed herein. For example, to improve a specified function of anantibody, a variant oligonucleotide library encoding for a portion ofthe antibody is designed and synthesized. A variant oligonucleotidelibrary for the antibody may then be generated by processes describedherein (e.g., PCR mutagenesis followed by insertion into a vector). Theantibody is then expressed in a production cell line and screened forenhanced activity. Example screens include examining modulation inbinding affinity to an antigen, stability, or effector function (e.g.,ADCC, complement, or apoptosis). Exemplary regions to optimize theantibody include, without limitation, the Fc region, Fab region,variable region of the Fab region, constant region of the Fab region,variable domain of the heavy chain or light chain (V_(H) or V_(L)), andspecific complementarity-determining regions (CDRs) of V_(H) or V_(L).

Nucleic acid libraries synthesized by methods described herein may beexpressed in various cells associated with a disease state. Cellsassociated with a disease state include cell lines, tissue samples,primary cells from a subject, cultured cells expanded from a subject, orcells in a model system. Exemplary cells include without limitation,prokaryotic and eukaryotic cells. Exemplary eukaryotic cells include,without limitation, animal, plant, and fungal cells. Exemplary animalcells include, without limitation, insect, fish and mammalian cells.Exemplary mammalian cells include mouse, human, and primate cells.Exemplary model systems include, without limitation, plant and animalmodels of a disease state. Exemplary animals include, withoutlimitation, mice, rabbits, primates, fish, and insects. Exemplary plantsinclude, without limitation, a monocot and dicot. Exemplary plants alsoinclude, without limitation, microalgae, kelp, cyanobacteria, and green,brown and red algae, wheat, tobacco, and corn, rice, cotton, vegetables,and fruit.

To identify a variant molecule associated with prevention, reduction ortreatment of a disease state, a variant nucleic acid library describedherein is expressed in a cell associated with a disease state, or one inwhich a cell a disease state may be induced. In some instances, an agentis used to induce a disease state in cells. Exemplary tools for diseasestate induction include, without limitation, a Cre/Lox recombinationsystem, LPS inflammation induction, and streptozotocin to inducehypoglycemia. The cells associated with a disease state may be cellsfrom a model system or cultured cells, as well as cells from a subjecthaving a particular disease condition. Exemplary disease conditionsinclude a bacterial, fungal, viral, autoimmune, or proliferativedisorder (e.g., cancer). In some instances, the variant nucleic acidlibrary is expressed in the model system, cell line, or primary cellsderived from a subject, and screened for changes in at least onecellular activity. Exemplary cellular activities include, withoutlimitation, proliferation, cycle progression, cell death, adhesion,migration, reproduction, cell signaling, energy production, oxygenutilization, metabolic activity, and aging, response to free radicaldamage, or any combination thereof.

Substrates

Provided herein are substrates comprising a plurality of clusters,wherein each cluster comprises a plurality of loci that support theattachment and synthesis of oligonucleotides. The term “locus” as usedherein refers to a discrete region on a structure which provides supportfor oligonucleotides encoding for a single predetermined sequence toextend from the surface. In some instances, a locus is on a twodimensional surface, e.g., a substantially planar surface. In someinstances, a locus refers to a discrete raised or lowered site on asurface e.g., a well, microwell, channel, or post. In some instances, asurface of a locus comprises a material that is actively functionalizedto attach to at least one nucleotide for oligonucleotide synthesis, orpreferably, a population of identical nucleotides for synthesis of apopulation of oligonucleotides. In some instances, oligonucleotiderefers to a population of oligonucleotides encoding for the same nucleicacid sequence. In some instances, a surface of a device is inclusive ofone or a plurality of surfaces of a substrate.

Average error rates for oligonucleotides synthesized within a libraryusing the systems and methods provided may be less than 1 in 1000, lessthan 1 in 1250, less than 1 in 1500, less than 1 in 2000, less than 1 in3000 or less often. In some instances, average error rates foroligonucleotides synthesized within a library using the systems andmethods provided are less than 1/500, 1/600, 1/700, 1/800, 1/900,1/1000, 1/1100, 1/1200, 1/1250, 1/1300, 1/1400, 1/1500, 1/1600, 1/1700,1/1800, 1/1900, 1/2000, 1/3000, or less. In some instances, averageerror rates for oligonucleotides synthesized within a library using thesystems and methods provided are less than 1/1000.

In some instances, aggregate error rates for oligonucleotidessynthesized within a library using the systems and methods provided areless than 1/500, 1/600, 1/700, 1/800, 1/900, 1/1000, 1/1100, 1/1200,1/1250, 1/1300, 1/1400, 1/1500, 1/1600, 1/1700, 1/1800, 1/1900, 1/2000,1/3000, or less compared to the predetermined sequences. In someinstances, aggregate error rates for oligonucleotides synthesized withina library using the systems and methods provided herein are less than1/500 or less compared to the predetermined sequences. In someinstances, aggregate error rates for oligonucleotides synthesized withina library using the systems and methods provided are less than 1/500,1/600, 1/700, 1/800, 1/900, or 1/1000. In some instances, aggregateerror rates for oligonucleotides synthesized within a library using thesystems and methods provided are less than 1/1000.

In some instances, an error correction enzyme may be used foroligonucleotides synthesized within a library using the systems andmethods provided herein. In some instances, aggregate error rates foroligonucleotides with error correction may be less than 1/500, 1/600,1/700, 1/800, 1/900, 1/1000, 1/1100, 1/1200, 1/1300, 1/1400, 1/1500,1/1600, 1/1700, 1/1800, 1/1900, 1/2000, 1/3000, or less compared to thepredetermined sequences. In some instances, aggregate error rates witherror correction for oligonucleotides synthesized within a library usingthe systems and methods provided may be less than 1/500, 1/600, 1/700,1/800, 1/900, or 1/1000. In some instances, aggregate error rates witherror correction for oligonucleotides synthesized within a library usingthe systems and methods provided may be less than 1/1000.

Error rate may limit the value of gene synthesis for the production oflibraries of gene variants. With an error rate of 1/300, about 0.7% ofthe clones in a 1500 base pair gene will be correct. As most of theerrors from oligonucleotide synthesis result in frame-shift mutations,over 99% of the clones in such a library will not produce a full-lengthprotein. Reducing the error rate by 75% would increase the fraction ofclones that are correct by a factor of 40. The methods and compositionsof the disclosure allow for fast de novo synthesis of largeoligonucleotide and gene libraries with error rates that are lower thancommonly observed gene synthesis methods both due to the improvedquality of synthesis and the applicability of error correction methodsthat are enabled in a massively parallel and time-efficient manner.Accordingly, libraries may be synthesized with base insertion, deletion,substitution, or total error rates that are under 1/300, 1/400, 1/500,1/600, 1/700, 1/800, 1/900, 1/1000, 1/1250, 1/1500, 1/2000, 1/2500,1/3000, 1/4000, 1/5000, 1/6000, 1/7000, 1/8000, 1/9000, 1/10000,1/12000, 1/15000, 1/20000, 1/25000, 1/30000, 1/40000, 1/50000, 1/60000,1/70000, 1/80000, 1/90000, 1/100000, 1/125000, 1/150000, 1/200000,1/300000, 1/400000, 1/500000, 1/600000, 1/700000, 1/800000, 1/900000,1/1000000, or less, across the library, or across more than 80%, 85%,90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 99.95%, 99.98%,99.99%, or more of the library. The methods and compositions of thedisclosure further relate to large synthetic oligonucleotide and genelibraries with low error rates associated with at least 30%, 40%, 50%,60%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.8%, 99.9%, 99.95%, 99.98%, 99.99%, or more of the oligonucleotides orgenes in at least a subset of the library to relate to error freesequences in comparison to a predetermined/preselected sequence. In someinstances, at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%,95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 99.95%, 99.98%, 99.99%, ormore of the oligonucleotides or genes in an isolated volume within thelibrary have the same sequence. In some instances, at least 30%, 40%,50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.8%, 99.9%, 99.95%, 99.98%, 99.99%, or more of any oligonucleotides orgenes related with more than 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%,99.7%, 99.8%, 99.9% or more similarity or identity have the samesequence. In some instances, the error rate related to a specified locuson an oligonucleotide or gene is optimized. Thus, a given locus or aplurality of selected loci of one or more oligonucleotides or genes aspart of a large library may each have an error rate that is less than1/300, 1/400, 1/500, 1/600, 1/700, 1/800, 1/900, 1/1000, 1/1250, 1/1500,1/2000, 1/2500, 1/3000, 1/4000, 1/5000, 1/6000, 1/7000, 1/8000, 1/9000,1/10000, 1/12000, 1/15000, 1/20000, 1/25000, 1/30000, 1/40000, 1/50000,1/60000, 1/70000, 1/80000, 1/90000, 1/100000, 1/125000, 1/150000,1/200000, 1/300000, 1/400000, 1/500000, 1/600000, 1/700000, 1/800000,1/900000, 1/1000000, or less. In various instances, such error optimizedloci may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000,4000, 5000, 6000, 7000, 8000, 9000, 10000, 30000, 50000, 75000, 100000,500000, 1000000, 2000000, 3000000 or more loci. The error optimized locimay be distributed to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500,3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 30000, 75000, 100000,500000, 1000000, 2000000, 3000000 or more oligonucleotides or genes.

The error rates may be achieved with or without error correction. Theerror rates may be achieved across the library, or across more than 80%,85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 99.95%,99.98%, 99.99%, or more of the library.

Provided herein are structures that may comprise a surface that supportsthe synthesis of a plurality of oligonucleotides having differentpredetermined sequences at addressable locations on a common support. Insome instances, a device provides support for the synthesis of more than2,000; 5,000; 10,000; 20,000; 30,000; 50,000; 75,000; 100,000; 200,000;300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000;1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000;2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000;10,000,000 or more non-identical oligonucleotides. In some instances,the device provides support for the synthesis of more than 2,000; 5,000;10,000; 20,000; 30,000; 50,000; 75,000; 100,000; 200,000; 300,000;400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000;1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000;3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 ormore oligonucleotides encoding for distinct sequences. In someinstances, at least a portion of the oligonucleotides have an identicalsequence or are configured to be synthesized with an identical sequence.

Provided herein are methods and devices for manufacture and growth ofoligonucleotides about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125,150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475,500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,1800, 1900, or 2000 bases in length. In some instances, the length ofthe oligonucleotide formed is about 5, 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 125, 150, 175, 200, or 225 bases in length. An oligonucleotidemay be at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 bases inlength. An oligonucleotide may be from 10 to 225 bases in length, from12 to 100 bases in length, from 20 to 150 bases in length, from 20 to130 bases in length, from 30 to 100 bases in length, or from 30 to 70bases in length.

In some instances, oligonucleotides are synthesized on distinct loci ofa substrate, wherein each locus supports the synthesis of a populationof oligonucleotides. In some instances, each locus supports thesynthesis of a population of oligonucleotides having a differentsequence than a population of oligonucleotides grown on another locus.In some instances, the loci of a device are located within a pluralityof clusters. In some instances, a device comprises at least 10, 500,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000,12000, 13000, 14000, 15000, 20000, 30000, 40000, 50000 or more clusters.In some instances, a device comprises more than 2,000; 5,000; 10,000;100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000;900,000; 1,000,000; 1,100,000; 1,200,000; 1,300,000; 1,400,000;1,500,000; 1,600,000; 1,700,000; 1,800,000; 1,900,000; 2,000,000;300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000;1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000;2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; or10,000,000 or more distinct loci. In some instances, a device comprisesabout 10,000 distinct loci. The amount of loci within a single clusteris varied in different instances. In some instances, each clusterincludes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 120, 130, 150, 200, 300, 400, 500 or more loci. In some instances,each cluster includes about 50-500 loci. In some instances, each clusterincludes about 100-200 loci. In some instances, each cluster includesabout 100-150 loci. In some instances, each cluster includes about 109,121, 130 or 137 loci. In some instances, each cluster includes about 19,20, 61, 64 or more loci.

The number of distinct oligonucleotides synthesized on a device may bedependent on the number of distinct loci available in the substrate. Insome instances, the density of loci within a cluster of a device is atleast or about 1 locus per mm², 10 loci per mm², 25 loci per mm², 50loci per mm², 65 loci per mm², 75 loci per mm², 100 loci per mm², 130loci per mm², 150 loci per mm², 175 loci per mm², 200 loci per mm², 300loci per mm², 400 loci per mm², 500 loci per mm², 1,000 loci per mm² ormore. In some instances, a device comprises from about 10 loci per mm²to about 500 mm², from about 25 loci per mm² to about 400 mm², fromabout 50 loci per mm² to about 500 mm², from about 100 loci per mm² toabout 500 mm², from about 150 loci per mm² to about 500 mm², from about10 loci per mm² to about 250 mm², from about 50 loci per mm² to about250 mm², from about 10 loci per mm² to about 200 mm², or from about 50loci per mm² to about 200 mm². In some instances, the distance from thecenters of two adjacent loci within a cluster is from about 10 um toabout 500 um, from about 10 um to about 200 um, or from about 10 um toabout 100 um. In some instances, the distance from two centers ofadjacent loci is greater than about 10 um, 20 um, 30 um, 40 um, 50 um,60 um, 70 um, 80 um, 90 um or 100 um. In some instances, the distancefrom the centers of two adjacent loci is less than about 200 um, 150 um,100 um, 80 um, 70 um, 60 um, 50 um, 40 um, 30 um, 20 um or 10 um. Insome instances, each locus has a width of about 0.5 um, 1 um, 2 um, 3um, 4 um, 5 um, 6 um, 7 um, 8 um, 9 um, 10 um, 20 um, 30 um, 40 um, 50um, 60 um, 70 um, 80 um, 90 um or 100 um. In some instances, each locushas a width of about 0.5 um to 100 um, about 0.5 um to 50 um, about 10um to 75 um, or about 0.5 um to 50 um.

In some instances, the density of clusters within a device is at leastor about 1 cluster per 100 mm², 1 cluster per 10 mm², 1 cluster per 5mm², 1 cluster per 4 mm², 1 cluster per 3 mm², 1 cluster per 2 mm², 1cluster per 1 mm², 2 clusters per 1 mm², 3 clusters per 1 mm², 4clusters per 1 mm², 5 clusters per 1 mm², 10 clusters per 1 mm², 50clusters per 1 mm² or more. In some instances, a device comprises fromabout 1 cluster per 10 mm² to about 10 clusters per 1 mm². In someinstances, the distance from the centers of two adjacent clusters isless than about 50 um, 100 um, 200 um, 500 um, 1000 um, or 2000 um or5000 um. In some instances, the distance from the centers of twoadjacent clusters is from about 50 um to about 100 um, from about 50 umto about 200 um, from about 50 um to about 300 um, from about 50 um toabout 500 um, and from about 100 um to about 2000 um. In some instances,the distance from the centers of two adjacent clusters is from about0.05 mm to about 50 mm, from about 0.05 mm to about 10 mm, from about0.05 mm to about 5 mm, from about 0.05 mm to about 4 mm, from about 0.05mm to about 3 mm, from about 0.05 mm to about 2 mm, from about 0.1 mm to10 mm, from about 0.2 mm to 10 mm, from about 0.3 mm to about 10 mm,from about 0.4 mm to about 10 mm, from about 0.5 mm to 10 mm, from about0.5 mm to about 5 mm, or from about 0.5 mm to about 2 mm. In someinstances, each cluster has a diameter or width along one dimension ofabout 0.5 to 2 mm, about 0.5 to 1 mm, or about 1 to 2 mm. In someinstances, each cluster has a diameter or width along one dimension ofabout 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9 or 2 mm. In some instances, each cluster has an interiordiameter or width along one dimension of about 0.5, 0.6, 0.7, 0.8, 0.9,1, 1.1, 1.15, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mm.

Structures for oligonucleotide synthesis for use with devices,compositions, systems, and methods as described herein comprise avariety of sizes. A device may be about the size of a standard 96 wellplate, for example from about 100 and 200 mm by about 50 and 150 mm. Insome instances, a device has a diameter less than or equal to about 1000mm, 500 mm, 450 mm, 400 mm, 300 mm, 250 nm, 200 mm, 150 mm, 100 mm or 50mm. In some instances, the diameter of a device is from about 25 mm to1000 mm, from about 25 mm to about 800 mm, from about 25 mm to about 600mm, from about 25 mm to about 500 mm, from about 25 mm to about 400 mm,from about 25 mm to about 300 mm, or from about 25 mm to about 200.Non-limiting examples of device size include about 300 mm, 200 mm, 150mm, 130 mm, 100 mm, 76 mm, 51 mm and 25 mm. In some instances, a devicehas a planar surface area of at least about 100 mm²; 200 mm²; 500 mm²;1,000 mm²; 2,000 mm²; 5,000 mm²; 10,000 mm²; 12,000 mm²; 15,000 mm²;20,000 mm²; 30,000 mm²; 40,000 mm²; 50,000 mm² or more. In someinstances, the thickness of a device is from about 50 mm to about 2000mm, from about 50 mm to about 1000 mm, from about 100 mm to about 1000mm, from about 200 mm to about 1000 mm, or from about 250 mm to about1000 mm. Non-limiting examples of device thickness include 275 mm, 375mm, 525 mm, 625 mm, 675 mm, 725 mm, 775 mm and 925 mm. In someinstances, the thickness of a device varies with diameter and depends onthe composition of the substrate. For example, a device comprisingmaterials other than silicon has a different thickness than a silicondevice of the same diameter. Device thickness may be determined by themechanical strength of the material used and the device must be thickenough to support its own weight without cracking during handling. Insome instances, a structure comprises a plurality of devices describedherein.

Surface Materials

Provided herein is a device comprising a surface, wherein the surface ismodified to support oligonucleotide synthesis at predetermined locationsand with a resulting low error rate, a low dropout rate, a high yield,and a high oligo representation. In some embodiments, surfaces of adevice for oligonucleotide synthesis provided herein are fabricated froma variety of materials capable of modification to support a de novooligonucleotide synthesis reaction. In some cases, the devices aresufficiently conductive, e.g., are able to form uniform electric fieldsacross all or a portion of the device. A device described herein maycomprise a flexible material. Exemplary flexible materials include,without limitation, modified nylon, unmodified nylon, nitrocellulose,and polypropylene. A device described herein may comprise a rigidmaterial. Exemplary rigid materials include, without limitation, glass,fuse silica, silicon, silicon dioxide, silicon nitride, plastics (forexample, polytetrafluoroethylene, polypropylene, polystyrene,polycarbonate, and blends thereof, and metals (for example, gold,platinum). Devices disclosed herein may be fabricated from a materialcomprising silicon, polystyrene, agarose, dextran, cellulosic polymers,polyacrylamides, polydimethylsiloxane (PDMS), glass, or any combinationthereof. In some cases, a device disclosed herein is manufactured with acombination of materials listed herein or any other suitable materialknown in the art.

In some cases, a device disclosed herein comprises a silicon dioxidebase and a surface layer of silicon oxide. Alternatively, the device mayhave a base of silicon oxide. A surface of the device provided here maybe textured, resulting in an increase in overall surface area foroligonucleotide synthesis. Devices disclosed herein may comprise atleast 5%, 10%, 25%, 50%, 80%, 90%, 95%, or 99% silicon. A devicedisclosed herein may be fabricated from a silicon on insulator (SOI)wafer.

Substrates, devices and reactors provided herein are fabricated from anyvariety of materials suitable for the methods and compositions describedherein. In certain instances, device materials are fabricated to exhibita low level of nucleotide binding. In some instances, device materialsare modified to generate distinct surfaces that exhibit a high level ofnucleotide binding. In some instances, device materials are transparentto visible and/or UV light. In some instances, device materials aresufficiently conductive, e.g., are able to form uniform electric fieldsacross all or a portion of a substrate. In some instances, conductivematerials are connected to an electric ground. In some instances, thedevice is heat conductive or insulated. In some instances, the materialsare chemical resistant and heat resistant to support chemical orbiochemical reactions, for example oligonucleotide synthesis reactionprocesses. In some instances, a device comprises flexible materials.Flexible materials include, without limitation, modified nylon,unmodified nylon, nitrocellulose, polypropylene, and the like. In someinstances, a device comprises rigid materials. Rigid materials include,without limitation, glass, fuse silica, silicon, silicon dioxide,silicon nitride, plastics (for example, polytetraflouroethylene,polypropylene, polystyrene, polycarbonate, and blends thereof, and thelike), and metals (for example, gold, platinum, and the like). In someinstances, a device is fabricated from a material comprising silicon,polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides,polydimethylsiloxane (PDMS), glass, or any combination thereof. In someinstances, a device is manufactured with a combination of materialslisted herein or any other suitable material known in the art.

Surface Architecture

Provided herein are devices comprising raised and/or lowered features.One benefit of having such features is an increase in surface area tosupport oligonucleotide synthesis. In some instances, a device havingraised and/or lowered features is referred to as a three-dimensionalsubstrate. In some instances, a three-dimensional device comprises oneor more channels. In some instances, one or more loci comprise achannel. In some instances, the channels are accessible to reagentdeposition via a deposition device such as an oligonucleotidesynthesizer. In some instances, reagents and/or fluids collect in alarger well in fluid communication with one or more channels. Forexample, a device comprises a plurality of channels corresponding to aplurality of loci with a cluster, and the plurality of channels are influid communication with one well of the cluster. In some methods, alibrary of oligonucleotides is synthesized in a plurality of loci of acluster.

In some instances, the structure is configured to allow for controlledflow and mass transfer paths for oligonucleotide synthesis on a surface.In some instances, the configuration of a device allows for thecontrolled and even distribution of mass transfer paths, chemicalexposure times, and/or wash efficacy during oligonucleotide synthesis.In some instances, the configuration of a device allows for increasedsweep efficiency, for example by providing sufficient volume for growingan oligonucleotide such that the excluded volume by the growingoligonucleotide does not take up more than 50, 45, 40, 35, 30, 25, 20,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1%, or less of theinitially available volume that is available or suitable for growing theoligonucleotide. In some instances, a three-dimensional structure allowsfor managed flow of fluid to allow for the rapid exchange of chemicalexposure.

Provided herein are methods to synthesize an amount of DNA of 1 fM, 5fM, 10 fM, 25 fM, 50 fM, 75 fM, 100 fM, 200 fM, 300 fM, 400 fM, 500 fM,600 fM, 700 fM, 800 fM, 900 fM, 1 pM, 5 pM, 10 pM, 25 pM, 50 pM, 75 pM,100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM,or more. In some instances, an oligonucleotide library may span thelength of about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, or 100% of a gene. A gene may be varied up to about1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%,90%, 95%, or 100%.

Non-identical oligonucleotides may collectively encode a sequence for atleast 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,85%, 90%, 95%, or 100% of a gene. In some instances, an oligonucleotidemay encode a sequence of 50%, 60%, 70%, 80%, 85%, 90%, 95%, or more of agene. In some instances, an oligonucleotide may encode a sequence of80%, 85%, 90%, 95%, or more of a gene.

In some instances, segregation is achieved by physical structure. Insome instances, segregation is achieved by differentialfunctionalization of the surface generating active and passive regionsfor oligonucleotide synthesis. Differential functionalization is alsoachieved by alternating the hydrophobicity across the device surface,thereby creating water contact angle effects that cause beading orwetting of the deposited reagents. Employing larger structures maydecrease splashing and cross-contamination of distinct oligonucleotidesynthesis locations with reagents of the neighboring spots. In someinstances, a device, such as an oligonucleotide synthesizer, is used todeposit reagents to distinct oligonucleotide synthesis locations.Substrates having three-dimensional features are configured in a mannerthat allows for the synthesis of a large number of oligonucleotides(e.g., more than about 10,000) with a low error rate (e.g., less thanabout 1:500, 1:1000, 1:1500, 1:2,000; 1:3,000; 1:5,000; or 1:10,000). Insome instances, a device comprises features with a density of about orgreater than about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 300, 400 or 500 features permm².

A well of a device may have the same or different width, height, and/orvolume as another well of the substrate. A channel of a device may havethe same or different width, height, and/or volume as another channel ofthe substrate. In some instances, the width of a cluster is from about0.05 mm to about 50 mm, from about 0.05 mm to about 10 mm, from about0.05 mm to about 5 mm, from about 0.05 mm to about 4 mm, from about 0.05mm to about 3 mm, from about 0.05 mm to about 2 mm, from about 0.05 mmto about 1 mm, from about 0.05 mm to about 0.5 mm, from about 0.05 mm toabout 0.1 mm, from about 0.1 mm to 10 mm, from about 0.2 mm to about 10mm, from about 0.3 mm to about 10 mm, from about 0.4 mm to about 10 mm,from about 0.5 mm to about 10 mm, from about 0.5 mm to about 5 mm, orfrom about 0.5 mm to about 2 mm. In some instances, the width of a wellcomprising a cluster is from about 0.05 mm to about 50 mm, from about0.05 mm to about 10 mm, from about 0.05 mm to about 5 mm, from about0.05 mm to about 4 mm, from about 0.05 mm to about 3 mm, from about 0.05mm to about 2 mm, from about 0.05 mm to about 1 mm, from about 0.05 mmto about 0.5 mm, from about 0.05 mm to about 0.1 mm, from about 0.1 mmto about 10 mm, from about 0.2 mm to about 10 mm, from about 0.3 mm toabout 10 mm, from about 0.4 mm to about 10 mm, from about 0.5 mm toabout 10 mm, from about 0.5 mm to about 5 mm, or from about 0.5 mm toabout 2 mm. In some instances, the width of a cluster is less than orabout 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, 0.5 mm, 0.1 mm, 0.09 mm, 0.08 mm,0.07 mm, 0.06 mm or 0.05 mm. In some instances, the width of a clusteris from about 1.0 to about 1.3 mm. In some instances, the width of acluster is about 1.150 mm. In some instances, the width of a well isless than or about 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, 0.5 mm, 0.1 mm, 0.09mm, 0.08 mm, 0.07 mm, 0.06 mm or 0.05 mm. In some instances, the widthof a well is from about 1.0 to about 1.3 mm. In some instances, thewidth of a well is about 1.150 mm. In some instances, the width of acluster is about 0.08 mm. In some instances, the width of a well isabout 0.08 mm. The width of a cluster may refer to clusters within atwo-dimensional or three-dimensional substrate.

In some instances, the height of a well is from about 20 um to about1000 um, from about 50 um to about 1000 um, from about 100 um to about1000 um, from about 200 um to about 1000 um, from about 300 um to about1000 um, from about 400 um to about 1000 um, or from about 500 um toabout 1000 um. In some instances, the height of a well is less thanabout 1000 um, less than about 900 um, less than about 800 um, less thanabout 700 um, or less than about 600 um.

In some instances, a device comprises a plurality of channelscorresponding to a plurality of loci within a cluster, wherein theheight or depth of a channel is from about 5 um to about 500 um, fromabout 5 um to about 400 um, from about 5 um to about 300 um, from about5 um to about 200 um, from about 5 um to about 100 um, from about 5 umto about 50 um, or from about 10 um to about 50 um. In some instances,the height of a channel is less than 100 um, less than 80 um, less than60 um, less than 40 um or less than 20 um.

In some instances, the diameter of a channel, locus (e.g., in asubstantially planar substrate) or both channel and locus (e.g., in athree-dimensional device wherein a locus corresponds to a channel) isfrom about 1 um to about 1000 um, from about 1 um to about 500 um, fromabout 1 um to about 200 um, from about 1 um to about 100 um, from about5 um to about 100 um, or from about 10 um to about 100 um, for example,about 90 um, 80 um, 70 um, 60 um, 50 um, 40 um, 30 um, 20 um or 10 um.In some instances, the diameter of a channel, locus, or both channel andlocus is less than about 100 um, 90 um, 80 um, 70 um, 60 um, 50 um, 40um, 30 um, 20 um or 10 um. In some instances, the distance from thecenter of two adjacent channels, loci, or channels and loci is fromabout 1 um to about 500 um, from about 1 um to about 200 um, from about1 um to about 100 um, from about 5 um to about 200 um, from about 5 umto about 100 um, from about 5 um to about 50 um, or from about 5 um toabout 30 um, for example, about 20 um.

Surface Modifications

In various instances, surface modifications are employed for thechemical and/or physical alteration of a surface by an additive orsubtractive process to change one or more chemical and/or physicalproperties of a device surface or a selected site or region of a devicesurface. For example, surface modifications include, without limitation,(1) changing the wetting properties of a surface, (2) functionalizing asurface, i.e., providing, modifying or substituting surface functionalgroups, (3) defunctionalizing a surface, i.e., removing surfacefunctional groups, (4) otherwise altering the chemical composition of asurface, e.g., through etching, (5) increasing or decreasing surfaceroughness, (6) providing a coating on a surface, e.g., a coating thatexhibits wetting properties that are different from the wettingproperties of the surface, and/or (7) depositing particulates on asurface.

In some instances, the addition of a chemical layer on top of a surface(referred to as an adhesion promoter) facilitates structured patterningof loci on a surface of a substrate. Exemplary surfaces for applicationof adhesion promotion include, without limitation, glass, silicon,silicon dioxide and silicon nitride. In some instances, the adhesionpromoter is a chemical with a high surface energy. In some instances, asecond chemical layer is deposited on a surface of a substrate. In someinstances, the second chemical layer has a low surface energy. In someinstances, surface energy of a chemical layer coated on a surfacesupports localization of droplets on the surface. Depending on thepatterning arrangement selected, the proximity of loci and/or area offluid contact at the loci are alterable.

In some instances, a device surface, or resolved loci, onto whichnucleic acids or other moieties are deposited, e.g., for oligonucleotidesynthesis, are smooth or substantially planar (e.g., two-dimensional) orhave irregularities, such as raised or lowered features (e.g.,three-dimensional features). In some instances, a device surface ismodified with one or more different layers of compounds. Suchmodification layers of interest include, without limitation, inorganicand organic layers such as metals, metal oxides, polymers, small organicmolecules and the like. Non-limiting polymeric layers include peptides,proteins, nucleic acids or mimetics thereof (e.g., peptide nucleic acidsand the like), polysaccharides, phospholipids, polyurethanes,polyesters, polycarbonates, polyureas, polyamides, polyetheyleneamines,polyarylene sulfides, polysiloxanes, polyimides, polyacetates, and anyother suitable compounds described herein or otherwise known in the art.In some instances, polymers are heteropolymeric. In some instances,polymers are homopolymeric. In some instances, polymers comprisefunctional moieties or are conjugated.

In some instances, resolved loci of a device are functionalized with oneor more moieties that increase and/or decrease surface energy. In someinstances, a moiety is chemically inert. In some instances, a moiety isconfigured to support a desired chemical reaction, for example, one ormore processes in an oligonucleotide synthesis reaction. The surfaceenergy, or hydrophobicity, of a surface is a factor for determining theaffinity of a nucleotide to attach onto the surface. In some instances,a method for device functionalization may comprise: (a) providing adevice having a surface that comprises silicon dioxide; and (b)silanizing the surface using, a suitable silanizing agent describedherein or otherwise known in the art, for example, an organofunctionalalkoxysilane molecule.

In some instances, the organofunctional alkoxysilane molecule comprisesdimethylchloro-octodecyl-silane, methyldichloro-octodecyl-silane,trichloro-octodecyl-silane, trimethyl-octodecyl-silane,triethyl-octodecyl-silane, or any combination thereof. In someinstances, a device surface comprises functionalized withpolyethylene/polypropylene (functionalized by gamma irradiation orchromic acid oxidation, and reduction to hydroxyalkyl surface), highlycrosslinked polystyrene-divinylbenzene (derivatized bychloromethylation, and aminated to benzylamine functional surface),nylon (the terminal aminohexyl groups are directly reactive), or etchedwith reduced polytetrafluoroethylene. Other methods and functionalizingagents are described in U.S. Pat. No. 5,474,796, which is hereinincorporated by reference in its entirety.

In some instances, a device surface is functionalized by contact with aderivatizing composition that contains a mixture of silanes, underreaction conditions effective to couple the silanes to the devicesurface, typically via reactive hydrophilic moieties present on thedevice surface. Silanization generally covers a surface throughself-assembly with organofunctional alkoxysilane molecules.

A variety of siloxane functionalizing reagents may further be used ascurrently known in the art, e.g., for lowering or increasing surfaceenergy. The organofunctional alkoxysilanes may be classified accordingto their organic functions.

Provided herein are devices that may contain patterning of agentscapable of coupling to a nucleoside. In some instances, a device may becoated with an active agent. In some instances, a device may be coatedwith a passive agent. Exemplary active agents for inclusion in coatingmaterials described herein include, without limitation,N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS),11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane,(3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane,3-glycidoxypropyltrimethoxysilane (GOPS), 3-iodo-propyltrimethoxysilane,butyl-aldehydr-trimethoxysilane, dimeric secondary aminoalkyl siloxanes,(3-aminopropyl)-diethoxy-methylsilane,(3-aminopropyl)-dimethyl-ethoxysilane, and(3-aminopropyl)-trimethoxysilane,(3-glycidoxypropyl)-dimethyl-ethoxysilane, glycidoxy-trimethoxysilane,(3-mercaptopropyl)-trimethoxysilane, 3-4epoxycyclohexyl-ethyltrimethoxysilane, and(3-mercaptopropyl)-methyl-dimethoxysilane, allyl trichlorochlorosilane,7-oct-1-enyl trichlorochlorosilane, or bis (3-trimethoxysilylpropyl)amine.

Exemplary passive agents for inclusion in a coating material describedherein include, without limitation, perfluorooctyltrichlorosilane;tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane; 1H, 1H, 2H,2H-fluorooctyltriethoxysilane (FOS);trichloro(1H,1H,2H,2H-perfluorooctyl)silane;tert-butyl-[5-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indol-1-yl]-dimethyl-silane;CYTOP™; Fluorinert™; perfluoroctyltrichlorosilane (PFOTCS);perfluorooctyldimethylchlorosilane (PFODCS);perfluorodecyltriethoxysilane (PFDTES);pentafluorophenyl-dimethylpropylchloro-silane (PFPTES);perfluorooctyltriethoxysilane; perfluorooctyltrimethoxysilane;octylchlorosilane; dimethylchloro-octodecyl-silane;methyldichloro-octodecyl-silane; trichloro-octodecyl-silane;trimethyl-octodecyl-silane; triethyl-octodecyl-silane; oroctadecyltrichlorosilane.

In some instances, a functionalization agent comprises a hydrocarbonsilane such as octadecyltrichlorosilane. In some instances, thefunctionalizing agent comprises 11-acetoxyundecyltriethoxysilane,n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane,(3-aminopropyl)triethoxysilane, glycidyloxypropyl/trimethoxysilane andN-(3-triethoxysilylpropyl)-4-hydroxybutyramide.

Oligonucleotide Synthesis

Methods of the current disclosure for oligonucleotide synthesis mayinclude processes involving phosphoramidite chemistry. In someinstances, oligonucleotide synthesis comprises coupling a base withphosphoramidite. Oligonucleotide synthesis may comprise coupling a baseby deposition of phosphoramidite under coupling conditions, wherein thesame base is optionally deposited with phosphoramidite more than once,i.e., double coupling. Oligonucleotide synthesis may comprise capping ofunreacted sites. In some instances, capping is optional. Oligonucleotidesynthesis may also comprise oxidation or an oxidation step or oxidationsteps. Oligonucleotide synthesis may comprise deblocking, detritylation,and sulfurization. In some instances, oligonucleotide synthesiscomprises either oxidation or sulfurization. In some instances, betweenone or each step during an oligonucleotide synthesis reaction, thedevice is washed, for example, using tetrazole or acetonitrile. Timeframes for any one step in a phosphoramidite synthesis method may beless than about 2 min, 1 min, 50 sec, 40 sec, 30 sec, 20 sec and 10 sec.

Oligonucleotide synthesis using a phosphoramidite method may comprise asubsequent addition of a phosphoramidite building block (e.g.,nucleoside phosphoramidite) to a growing oligonucleotide chain for theformation of a phosphite triester linkage. Phosphoramiditeoligonucleotide synthesis proceeds in the 3′ to 5′ direction.Phosphoramidite oligonucleotide synthesis allows for the controlledaddition of one nucleotide to a growing nucleic acid chain per synthesiscycle. In some instances, each synthesis cycle comprises a couplingstep. Phosphoramidite coupling involves the formation of a phosphitetriester linkage between an activated nucleoside phosphoramidite and anucleoside bound to the substrate, for example, via a linker. In someinstances, the nucleoside phosphoramidite is provided to the deviceactivated. In some instances, the nucleoside phosphoramidite is providedto the device with an activator. In some instances, nucleosidephosphoramidites are provided to the device in a 1.5, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50,60, 70, 80, 90, 100-fold excess or more over the substrate-boundnucleosides. In some instances, the addition of nucleosidephosphoramidite is performed in an anhydrous environment, for example,in anhydrous acetonitrile. Following addition of a nucleosidephosphoramidite, the device is optionally washed. In some instances, thecoupling step is repeated one or more additional times, optionally witha wash step between nucleoside phosphoramidite additions to thesubstrate. In some instances, an oligonucleotide synthesis method usedherein comprises 1, 2, 3 or more sequential coupling steps. Prior tocoupling, in many cases, the nucleoside bound to the device isde-protected by removal of a protecting group, where the protectinggroup functions to prevent polymerization. A common protecting group is4,4′-dimethoxytrityl (DMT).

Following coupling, phosphoramidite oligonucleotide synthesis methodsoptionally comprise a capping step. In a capping step, the growingoligonucleotide is treated with a capping agent. A capping step isuseful to block unreacted substrate-bound 5′-OH groups after couplingfrom further chain elongation, preventing the formation ofoligonucleotides with internal base deletions. Further, phosphoramiditesactivated with 1H-tetrazole may react, to a small extent, with the O6position of guanosine. Without being bound by theory, upon oxidationwith I₂/water, this side product, possibly via O6-N7 migration, mayundergo depurination. The apurinic sites may end up being cleaved in thecourse of the final deprotection of the oligonucleotide thus reducingthe yield of the full-length product. The O6 modifications may beremoved by treatment with the capping reagent prior to oxidation withI₂/water. In some instances, inclusion of a capping step duringoligonucleotide synthesis decreases the error rate as compared tosynthesis without capping. As an example, the capping step comprisestreating the substrate-bound oligonucleotide with a mixture of aceticanhydride and 1-methylimidazole. Following a capping step, the device isoptionally washed.

In some instances, following addition of a nucleoside phosphoramidite,and optionally after capping and one or more wash steps, the devicebound growing oligonucleotide is oxidized. The oxidation step comprisesthe phosphite triester is oxidized into a tetracoordinated phosphatetriester, a protected precursor of the naturally occurring phosphatediester internucleoside linkage. In some instances, oxidation of thegrowing oligonucleotide is achieved by treatment with iodine and water,optionally in the presence of a weak base (e.g., pyridine, lutidine,collidine). Oxidation may be carried out under anhydrous conditionsusing, e.g. tert-Butyl hydroperoxide or(1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). In some methods, acapping step is performed following oxidation. A second capping stepallows for device drying, as residual water from oxidation that maypersist may inhibit subsequent coupling. Following oxidation, the deviceand growing oligonucleotide is optionally washed. In some instances, thestep of oxidation is substituted with a sulfurization step to obtainoligonucleotide phosphorothioates, wherein any capping steps may beperformed after the sulfurization. Many reagents are capable of theefficient sulfur transfer, including but not limited to3-(Dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT,3H-1,2-benzodithiol-3-one 1,1-dioxide, also known as Beaucage reagent,and N,N,N′N′-Tetraethylthiuram disulfide (TETD).

In order for a subsequent cycle of nucleoside incorporation to occurthrough coupling, the protected 5′ end of the device bound growingoligonucleotide is removed so that the primary hydroxyl group isreactive with a next nucleoside phosphoramidite. In some instances, theprotecting group is DMT and deblocking occurs with trichloroacetic acidin dichloromethane. Conducting detritylation for an extended time orwith stronger than recommended solutions of acids may lead to increaseddepurination of solid support-bound oligonucleotide and thus reduces theyield of the desired full-length product. Methods and compositions ofthe disclosure described herein provide for controlled deblockingconditions limiting undesired depurination reactions. In some instances,the device bound oligonucleotide is washed after deblocking. In someinstances, efficient washing after deblocking contributes to synthesizedoligonucleotides having a low error rate.

Methods for the synthesis of oligonucleotides typically involve aniterating sequence of the following steps: application of a protectedmonomer to an actively functionalized surface (e.g., locus) to link witheither the activated surface, a linker or with a previously deprotectedmonomer; deprotection of the applied monomer so that it is reactive witha subsequently applied protected monomer; and application of anotherprotected monomer for linking. One or more intermediate steps includeoxidation or sulfurization. In some instances, one or more wash stepsprecede or follow one or all of the steps.

Methods for phosphoramidite-based oligonucleotide synthesis comprise aseries of chemical steps. In some instances, one or more steps of asynthesis method involve reagent cycling, where one or more steps of themethod comprise application to the device of a reagent useful for thestep. For example, reagents are cycled by a series of liquid depositionand vacuum drying steps. For substrates comprising three-dimensionalfeatures such as wells, microwells, channels and the like, reagents areoptionally passed through one or more regions of the device via thewells and/or channels.

Methods and systems described herein relate to oligonucleotide synthesisdevices for the synthesis of oligonucleotides. The synthesis may be inparallel. For example, at least or about at least 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 1000, 10000, 50000, 75000, 100000 or moreoligonucleotides may be synthesized in parallel. The total numberoligonucleotides that may be synthesized in parallel may be from2-100000, 3-50000, 4-10000, 5-1000, 6-900, 7-850, 8-800, 9-750, 10-700,11-650, 12-600, 13-550, 14-500, 15-450, 16-400, 17-350, 18-300, 19-250,20-200, 21-150,22-100, 23-50, 24-45, 25-40, 30-35. Those of skill in theart appreciate that the total number of oligonucleotides synthesized inparallel may fall within any range bound by any of these values, forexample 25-100. The total number of oligonucleotides synthesized inparallel may fall within any range defined by any of the values servingas endpoints of the range. Total molar mass of oligonucleotidessynthesized within the device or the molar mass of each of theoligonucleotides may be at least or at least about 10, 20, 30, 40, 50,100, 250, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10000, 25000, 50000, 75000, 100000 picomoles, or more. The lengthof each of the oligonucleotides or average length of theoligonucleotides within the device may be at least or about at least 10,15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 300, 400, 500nucleotides, or more. The length of each of the oligonucleotides oraverage length of the oligonucleotides within the device may be at mostor about at most 500, 400, 300, 200, 150, 100, 50, 45, 35, 30, 25, 20,19, 18, 17, 16, 15, 14, 13, 12, 11, 10 nucleotides, or less. The lengthof each of the oligonucleotides or average length of theoligonucleotides within the device may fall from 10-500, 9-400, 11-300,12-200, 13-150, 14-100, 15-50, 16-45, 17-40, 18-35, 19-25. Those ofskill in the art appreciate that the length of each of theoligonucleotides or average length of the oligonucleotides within thedevice may fall within any range bound by any of these values, forexample 100-300. The length of each of the oligonucleotides or averagelength of the oligonucleotides within the device may fall within anyrange defined by any of the values serving as endpoints of the range.

Methods for oligonucleotide synthesis on a surface provided herein allowfor synthesis at a fast rate. As an example, at least 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175,200 nucleotides per hour, or more are synthesized. Nucleotides includeadenine, guanine, thymine, cytosine, uridine building blocks, oranalogs/modified versions thereof. In some instances, libraries ofoligonucleotides are synthesized in parallel on a substrate. Forexample, a device comprising about or at least about 100; 1,000; 10,000;30,000; 75,000; 100,000; 1,000,000; 2,000,000; 3,000,000; 4,000,000; or5,000,000 resolved loci is able to support the synthesis of at least thesame number of distinct oligonucleotides, wherein each oligonucleotideencoding a distinct sequence is synthesized on a resolved locus. In someinstances, a library of oligonucleotides is synthesized on a device withlow error rates described herein in less than about three months, twomonths, one month, three weeks, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2 days, 24 hours or less. In some instances, larger nucleic acidsassembled from an oligonucleotide library synthesized with a low errorrate using the substrates and methods described herein are prepared inless than about three months, two months, one month, three weeks, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, 24 hours or less.

In some instances, methods described herein provide for generation of alibrary of oligonucleotides comprising variant oligonucleotidesdiffering at a plurality of codon sites. In some instances, anoligonucleotide may have 1 site, 2 sites, 3 sites, 4 sites, 5 sites, 6sites, 7 sites, 8 sites, 9 sites, 10 sites, 11 sites, 12 sites, 13sites, 14 sites, 15 sites, 16 sites, 17 sites 18 sites, 19 sites, 20sites, 30 sites, 40 sites, 50 sites, or more of variant codon sites.

In some instances, the one or more sites of variant codon sites may beadjacent. In some instances, the one or more sites of variant codonsites may not be adjacent and separated by 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more codons.

In some instances, an oligonucleotide may comprise multiple sites ofvariant codon sites, wherein all the variant codon sites are adjacent toone another, forming a stretch of variant codon sites. In someinstances, an oligonucleotide may comprise multiple sites of variantcodon sites, wherein none the variant codon sites are adjacent to oneanother. In some instances, an oligonucleotide may comprise multiplesites of variant codon sites, wherein some the variant codon sites areadjacent to one another, forming a stretch of variant codon sites, andsome of the variant codon sites are not adjacent to one another.

Referring to the Figures, FIG. 12 illustrates an exemplary processworkflow for synthesis of nucleic acids (e.g., genes) from shorteroligonucleotides. The workflow is divided generally into phases: (1) denovo synthesis of a single stranded oligonucleotide library, (2) joiningoligonucleotides to form larger fragments, (3) error correction, (4)quality control, and (5) shipment. Prior to de novo synthesis, anintended nucleic acid sequence or group of nucleic acid sequences ispreselected. For example, a group of genes is preselected forgeneration.

Once large oligonucleotides for generation are selected, a predeterminedlibrary of oligonucleotides is designed for de novo synthesis. Varioussuitable methods are known for generating high density oligonucleotidearrays. In the workflow example, a device surface layer 1201 isprovided. In the example, chemistry of the surface is altered in orderto improve the oligonucleotide synthesis process. Areas of low surfaceenergy are generated to repel liquid while areas of high surface energyare generated to attract liquids. The surface itself may be in the formof a planar surface or contain variations in shape, such as protrusionsor microwells which increase surface area. In the workflow example, highsurface energy molecules selected serve a dual function of supportingDNA chemistry, as disclosed in International Patent ApplicationPublication WO/2015/021080, which is herein incorporated by reference inits entirety.

In situ preparation of oligonucleotide arrays is generated on a solidsupport and utilizes single nucleotide extension process to extendmultiple oligomers in parallel. A material deposition device, such as anoligonucleotide synthesizer, is designed to release reagents in a stepwise fashion such that multiple oligonucleotides extend, in parallel,one residue at a time to generate oligomers with a predetermined nucleicacid sequence 1202. In some instances, oligonucleotides are cleaved fromthe surface at this stage. Cleavage includes gas cleavage, e.g., withammonia or methylamine.

The generated oligonucleotide libraries are placed in a reactionchamber. In this exemplary workflow, the reaction chamber (also referredto as “nanoreactor”) is a silicon coated well, containing PCR reagentsand lowered onto the oligonucleotide library 1203. Prior to or after thesealing 1204 of the oligonucleotides, a reagent is added to release theoligonucleotides from the substrate. In the exemplary workflow, theoligonucleotides are released subsequent to sealing of the nanoreactor1205. Once released, fragments of single stranded oligonucleotideshybridize in order to span an entire long range sequence of DNA. Partialhybridization 1205 is possible because each synthesized oligonucleotideis designed to have a small portion overlapping with at least one otheroligonucleotide in the pool.

After hybridization, a PCA reaction is commenced. During the polymerasecycles, the oligonucleotides anneal to complementary fragments and gapsare filled in by a polymerase. Each cycle increases the length ofvarious fragments randomly depending on which oligonucleotides find eachother. Complementarity amongst the fragments allows for forming acomplete large span of double stranded DNA 1206.

After PCA is complete, the nanoreactor is separated from the device 1207and positioned for interaction with a device having primers for PCR1208. After sealing, the nanoreactor is subject to PCR 1209 and thelarger nucleic acids are amplified. After PCR 1210, the nanochamber isopened 1211, error correction reagents are added 1212, the chamber issealed 1213 and an error correction reaction occurs to remove mismatchedbase pairs and/or strands with poor complementarity from the doublestranded PCR amplification products 1214. The nanoreactor is opened andseparated 1215. Error corrected product is next subject to additionalprocessing steps, such as PCR and molecular bar coding, and thenpackaged 1222 for shipment 1223.

In some instances, quality control measures are taken. After errorcorrection, quality control steps include for example interaction with awafer having sequencing primers for amplification of the error correctedproduct 1216, sealing the wafer to a chamber containing error correctedamplification product 1217, and performing an additional round ofamplification 1218. The nanoreactor is opened 1219 and the products arepooled 1220 and sequenced 1221. After an acceptable quality controldetermination is made, the packaged product 1222 is approved forshipment 1223.

In some instances, a nucleic acid generated by a workflow such as thatin FIG. 12 is subject to mutagenesis using overlapping primers disclosedherein. In some instances, a library of primers is generated by in situpreparation on a solid support and utilize single nucleotide extensionprocess to extend multiple oligomers in parallel. A deposition device,such as an oligonucleotide synthesizer, is designed to release reagentsin a step wise fashion such that multiple oligonucleotides extend, inparallel, one residue at a time to generate oligomers with apredetermined nucleic acid sequence 1202.

Computer Systems

Any of the systems described herein, may be operably linked to acomputer and may be automated through a computer either locally orremotely. In various instances, the methods and systems of thedisclosure may further comprise software programs on computer systemsand use thereof. Accordingly, computerized control for thesynchronization of the dispense/vacuum/refill functions such asorchestrating and synchronizing the material deposition device movement,dispense action and vacuum actuation are within the bounds of thedisclosure. The computer systems may be programmed to interface betweenthe user specified base sequence and the position of a materialdeposition device to deliver the correct reagents to specified regionsof the substrate.

The computer system 1300 illustrated in FIG. 13 may be understood as alogical apparatus that can read instructions from media 1311 and/or anetwork port 1305, which can optionally be connected to server 1309having fixed media 1312. The system, such as shown in FIG. 13 caninclude a CPU 1301, disk drives 1303, optional input devices such askeyboard 1315 and/or mouse 1316 and optional monitor 1307. Datacommunication may be achieved through the indicated communication mediumto a server at a local or a remote location. The communication mediummay include any means of transmitting and/or receiving data. Forexample, the communication medium may be a network connection, awireless connection or an internet connection. Such a connection mayprovide for communication over the World Wide Web. It is envisioned thatdata relating to the present disclosure may be transmitted over suchnetworks or connections for reception and/or review by a party 1322 asillustrated in FIG. 13.

FIG. 14 is a block diagram illustrating a first example architecture ofa computer system 1400 that may be used in connection with exampleinstances of the present disclosure. As depicted in FIG. 14, the examplecomputer system may include a processor 1402 for processinginstructions. Non-limiting examples of processors include: Intel Xeon™processor, AMD Opteron™ processor, Samsung 32-bit RISC ARM 1176JZ(F)-Sv1.0™ processor, ARM Cortex-A8 Samsung S5PC100™ processor, ARM Cortex-A8Apple A4™ processor, Marvell PXA 930™ processor, or afunctionally-equivalent processor. Multiple threads of execution may beused for parallel processing. In some instances, multiple processors orprocessors with multiple cores may also be used, whether in a singlecomputer system, in a cluster, or distributed across systems over anetwork comprising a plurality of computers, cell phones, and/orpersonal data assistant devices.

As illustrated in FIG. 14, a high speed cache 1404 may be connected to,or incorporated in, the processor 1402 to provide a high speed memoryfor instructions or data that have been recently, or are frequently,used by processor 1402. The processor 1402 is connected to a northbridge 1406 by a processor bus 1408. The north bridge 1406 is connectedto random access memory (RAM) 1410 by a memory bus 1412 and managesaccess to the RAM 1410 by the processor 1402. The north bridge 1406 isalso connected to a south bridge 1414 by a chipset bus 1416. The southbridge 1414 is, in turn, connected to a peripheral bus 1418. Theperipheral bus may be, for example, PCI, PCI-X, PCI Express, or otherperipheral bus. The north bridge and south bridge are often referred toas a processor chipset and manage data transfer between the processor,RAM, and peripheral components on the peripheral bus 1418. In somealternative architectures, the functionality of the north bridge may beincorporated into the processor instead of using a separate north bridgechip. In some instances, system 1400 may include an accelerator card1422 attached to the peripheral bus 1418. The accelerator may includefield programmable gate arrays (FPGAs) or other hardware foraccelerating certain processing. For example, an accelerator may be usedfor adaptive data restructuring or to evaluate algebraic expressionsused in extended set processing.

Software and data are stored in external storage 1424 and may be loadedinto RAM 1410 and/or cache 1404 for use by the processor. The system1400 includes an operating system for managing system resources;non-limiting examples of operating systems include: Linux, Windows™,MACOS™, BlackBerry OS™, iOS™, and other functionally-equivalentoperating systems, as well as application software running on top of theoperating system for managing data storage and optimization inaccordance with example instances of the present disclosure. In thisexample, system 1400 also includes network interface cards (NICs) 1420and 1421 connected to the peripheral bus for providing networkinterfaces to external storage, such as Network Attached Storage (NAS)and other computer systems that may be used for distributed parallelprocessing.

FIG. 15 is a diagram showing a network 1500 with a plurality of computersystems 1502 a, and 1502 b, a plurality of cell phones and personal dataassistants 1502 c, and Network Attached Storage (NAS) 1504 a, and 1504b. In example instances, systems 1502 a, 1502 b, and 1502 c may managedata storage and optimize data access for data stored in NetworkAttached Storage (NAS) 1504 a and 1504 b. A mathematical model may beused for the data and be evaluated using distributed parallel processingacross computer systems 1502 a, and 1502 b, and cell phone and personaldata assistant systems 1502 c. Computer systems 1502 a, and 1502 b, andcell phone and personal data assistant systems 1502 c may also provideparallel processing for adaptive data restructuring of the data storedin Network Attached Storage (NAS) 1504 a and 1504 b. FIG. 15 illustratesan example only, and a wide variety of other computer architectures andsystems may be used in conjunction with the various instances of thepresent disclosure. For example, a blade server may be used to provideparallel processing. Processor blades may be connected through a backplane to provide parallel processing. Storage may also be connected tothe back plane or as Network Attached Storage (NAS) through a separatenetwork interface. In some example instances, processors may maintainseparate memory spaces and transmit data through network interfaces,back plane or other connectors for parallel processing by otherprocessors. In other instances, some or all of the processors may use ashared virtual address memory space.

FIG. 16 is a block diagram of a multiprocessor computer system 1600using a shared virtual address memory space in accordance with anexample instance. The system includes a plurality of processors 1602 a-fthat may access a shared memory subsystem 1604. The system incorporatesa plurality of programmable hardware memory algorithm processors (MAPs)1606 a-fin the memory subsystem 1604. Each MAP 1606 a-f may comprise amemory 1608 a-f and one or more field programmable gate arrays (FPGAs)1610 a-f. The MAP provides a configurable functional unit and particularalgorithms or portions of algorithms may be provided to the FPGAs 1610a-f for processing in close coordination with a respective processor.For example, the MAPs may be used to evaluate algebraic expressionsregarding the data model and to perform adaptive data restructuring inexample instances. In this example, each MAP is globally accessible byall of the processors for these purposes. In one configuration, each MAPmay use Direct Memory Access (DMA) to access an associated memory 1608a-f, allowing it to execute tasks independently of, and asynchronouslyfrom the respective microprocessor 1602 a-f. In this configuration, aMAP may feed results directly to another MAP for pipelining and parallelexecution of algorithms.

The above computer architectures and systems are examples only, and awide variety of other computer, cell phone, and personal data assistantarchitectures and systems may be used in connection with exampleinstances, including systems using any combination of generalprocessors, co-processors, FPGAs and other programmable logic devices,system on chips (SOCs), application specific integrated circuits(ASICs), and other processing and logic elements. In some instances, allor part of the computer system may be implemented in software orhardware. Any variety of data storage media may be used in connectionwith example instances, including random access memory, hard drives,flash memory, tape drives, disk arrays, Network Attached Storage (NAS)and other local or distributed data storage devices and systems.

In example instances, the computer system may be implemented usingsoftware modules executing on any of the above or other computerarchitectures and systems. In other instances, the functions of thesystem may be implemented partially or completely in firmware,programmable logic devices such as field programmable gate arrays(FPGAs) as referenced in FIG. 16, system on chips (SOCs), applicationspecific integrated circuits (ASICs), or other processing and logicelements. For example, the Set Processor and Optimizer may beimplemented with hardware acceleration through the use of a hardwareaccelerator card, such as accelerator card 1422 illustrated in FIG. 14.

The following examples are set forth to illustrate more clearly theprinciple and practice of embodiments disclosed herein to those skilledin the art and are not to be construed as limiting the scope of anyclaimed embodiments. Unless otherwise stated, all parts and percentagesare on a weight basis.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the disclosure and are not meant to limit the presentdisclosure in any fashion. The present examples, along with the methodsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of thedisclosure. Changes therein and other uses which are encompassed withinthe spirit of the disclosure as defined by the scope of the claims willoccur to those skilled in the art.

Example 1: Functionalization of a Device Surface

A device was functionalized to support the attachment and synthesis of alibrary of oligonucleotides. The device surface was first wet cleanedusing a piranha solution comprising 90% H₂SO₄ and 10% H₂O₂ for 20minutes. The device was rinsed in several beakers with DI water, heldunder a DI water gooseneck faucet for 5 min, and dried with N₂. Thedevice was subsequently soaked in NH₄OH (1:100; 3 mL:300 mL) for 5 min,rinsed with DI water using a handgun, soaked in three successive beakerswith DI water for 1 min each, and then rinsed again with DI water usingthe handgun. The device was then plasma cleaned by exposing the devicesurface to O₂. A SAMCO PC-300 instrument was used to plasma etch O₂ at250 watts for 1 min in downstream mode.

The cleaned device surface was actively functionalized with a solutioncomprising N-(3-triethoxysilylpropyl)-4-hydroxybutyramide using aYES-1224P vapor deposition oven system with the following parameters:0.5 to 1 torr, 60 min, 70° C., 135° C. vaporizer. The device surface wasresist coated using a Brewer Science 200X spin coater. SPR™ 3612photoresist was spin coated on the device at 2500 rpm for 40 sec. Thedevice was pre-baked for 30 min at 90° C. on a Brewer hot plate. Thedevice was subjected to photolithography using a Karl Suss MA6 maskaligner instrument. The device was exposed for 2.2 sec and developed for1 min in MSF 26A. Remaining developer was rinsed with the handgun andthe device soaked in water for 5 min. The device was baked for 30 min at100° C. in the oven, followed by visual inspection for lithographydefects using a Nikon L200. A descum process was used to remove residualresist using the SAMCO PC-300 instrument to O₂ plasma etch at 250 wattsfor 1 min.

The device surface was passively functionalized with a 100 μL solutionof perfluorooctyltrichlorosilane mixed with 10 μL light mineral oil. Thedevice was placed in a chamber, pumped for 10 min, and then the valvewas closed to the pump and left to stand for 10 min. The chamber wasvented to air. The device was resist stripped by performing two soaksfor 5 min in 500 mL NMP at 70° C. with ultrasonication at maximum power(9 on Crest system). The device was then soaked for 5 min in 500 mLisopropanol at room temperature with ultrasonication at maximum power.The device was dipped in 300 mL of 200 proof ethanol and blown dry withN₂. The functionalized surface was activated to serve as a support foroligonucleotide synthesis.

Example 2: Synthesis of a 50-mer Sequence on an OligonucleotideSynthesis Device

A two dimensional oligonucleotide synthesis device was assembled into aflowcell, which was connected to a flowcell (Applied Biosystems (ABI394DNA Synthesizer”). The two-dimensional oligonucleotide synthesis devicewas uniformly functionalized withN-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE (Gelest) was used tosynthesize an exemplary oligonucleotide of 50 bp (“50-meroligonucleotide”) using oligonucleotide synthesis methods describedherein.

The sequence of the 50-mer was as described in SEQ ID NO.: 20.5′AGACAATCAACCATTTGGGGTGGACAGCCTTGACCTCTAGACTTCGGCAT##TTTTTTT TTT3′ (SEQID NO.: 20), where # denotes Thymidine-succinyl hexamide CEDphosphoramidite (CLP-2244 from ChemGenes), which is a cleavable linkerenabling the release of oligos from the surface during deprotection.

The synthesis was done using standard DNA synthesis chemistry (coupling,capping, oxidation, and deblocking) according to the protocol in Table 5and an ABI synthesizer.

TABLE 5 Synthesis Protocol General DNA Synthesis Table 5 Process NameProcess Step Time (sec) WASH (Acetonitrile Acetonitrile System Flush 4Wash Flow) Acetonitrile to Flowcell 23 N2 System Flush 4 AcetonitrileSystem Flush 4 DNA BASE ADDITION Activator Manifold Flush 2(Phosphoramidite + Activator to Flowcell 6 Activator Flow) Activator + 6Phosphoramidite to Flowcell Activator to Flowcell 0.5 Activator + 5Phosphoramidite to Flowcell Activator to Flowcell 0.5 Activator + 5Phosphoramidite to Flowcell Activator to Flowcell 0.5 Activator + 5Phosphoramidite to Flowcell Incubate for 25 sec 25 WASH (AcetonitrileAcetonitrile System Flush 4 Wash Flow) Acetonitrile to Flowcell 15 N2System Flush 4 Acetonitrile System Flush 4 DNA BASE ADDITION ActivatorManifold Flush 2 (Phosphoramidite + Activator to Flowcell 5 ActivatorFlow) Activator + 18 Phosphoramidite to Flowcell Incubate for 25 sec 25WASH (Acetonitrile Acetonitrile System Flush 4 Wash Flow) Acetonitrileto Flowcell 15 N2 System Flush 4 Acetonitrile System Flush 4 CAPPING(CapA + B, CapA + B to Flowcell 15 1:1, Flow) WASH (AcetonitrileAcetonitrile System Flush 4 Wash Flow) Acetonitrile to Flowcell 15Acetonitrile System Flush 4 OXIDATION (Oxidizer Oxidizer to Flowcell 18Flow) WASH (Acetonitrile Acetonitrile System Flush 4 Wash Flow) N2System Flush 4 Acetonitrile System Flush 4 Acetonitrile to Flowcell 15Acetonitrile System Flush 4 Acetonitrile to Flowcell 15 N2 System Flush4 Acetonitrile System Flush 4 Acetonitrile to Flowcell 23 N2 SystemFlush 4 Acetonitrile System Flush 4 DEBLOCKING (Deblock Deblock toFlowcell 36 Flow) WASH (Acetonitrile Acetonitrile System Flush 4 WashFlow) N2 System Flush 4 Acetonitrile System Flush 4 Acetonitrile toFlowcell 18 N2 System Flush 4.13 Acetonitrile System Flush 4.13Acetonitrile to Flowcell 15

The phosphoramidite/activator combination was delivered similar to thedelivery of bulk reagents through the flowcell. No drying steps wereperformed as the environment stays “wet” with reagent the entire time.

The flow restrictor was removed from the ABI 394 synthesizer to enablefaster flow. Without flow restrictor, flow rates for amidites (0.1M inACN), Activator, (0.25M Benzoylthiotetrazole (“BTT”; 30-3070-xx fromGlenResearch) in ACN), and Ox (0.02M I2 in 20% pyridine, 10% water, and70% THF) were roughly ˜100 uL/sec, for acetonitrile (“ACN”) and cappingreagents (1:1 mix of CapA and CapB, wherein CapA is acetic anhydride inTHF/Pyridine and CapB is 16% 1-methylimidizole in THF), roughly ˜200uL/sec, and for Deblock (3% dichloroacetic acid in toluene), roughly˜300 uL/sec (compared to ˜50 uL/sec for all reagents with flowrestrictor). The time to completely push out Oxidizer was observed, thetiming for chemical flow times was adjusted accordingly and an extra ACNwash was introduced between different chemicals. After oligonucleotidesynthesis, the chip was deprotected in gaseous ammonia overnight at 75psi. Five drops of water were applied to the surface to recoveroligonucleotides. The recovered oligonucleotides were then analyzed on aBioAnalyzer small RNA chip (data not shown).

Example 3: Synthesis of a 100-mer Sequence on an OligonucleotideSynthesis Device

The same process as described in Example 2 for the synthesis of the50-mer sequence was used for the synthesis of a 100-mer oligonucleotide(“100-mer oligonucleotide”; 5′CGGGATCCTTATCGTCATCGTCGTACAGATCCCGACCCATTTGCTGTCCACCAGTCATGCTAGCCATACCATGATGATGATGATGATGAGAACCCCGCAT##TTTTTTTTTT3′, where # denotesThymidine-succinyl hexamide CED phosphoramidite (CLP-2244 fromChemGenes); SEQ ID NO.: 21) on two different silicon chips, the firstone uniformly functionalized withN-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE and the second onefunctionalized with 5/95 mix of 11-acetoxyundecyltriethoxysilane andn-decyltriethoxysilane, and the oligonucleotides extracted from thesurface were analyzed on a BioAnalyzer instrument (data not shown).

All ten samples from the two chips were further PCR amplified using aforward (5′ATGCGGGGTTCTCATCATC3′; SEQ ID NO.: 22) and a reverse(5′CGGGATCCTTATCGTCATCG3; SEQ ID NO.: 23) primer in a 50 uL PCR mix (25uL NEB Q5 mastermix, 2.5 uL 10 uM Forward primer, 2.5 uL 10 uM Reverseprimer, 1 uL oligonucleotide extracted from the surface, and water up to50 uL) using the following thermalcycling program:

-   -   98° C., 30 sec    -   98° C., 10 sec; 63° C., 10 sec; 72° C., 10 sec; repeat 12 cycles    -   72° C., 2 min

The PCR products were also run on a BioAnalyzer (data not shown),demonstrating sharp peaks at the 100-mer position. Next, the PCRamplified samples were cloned, and Sanger sequenced. Table 6 summarizesthe results from the Sanger sequencing for samples taken from spots 1-5from chip 1 and for samples taken from spots 6-10 from chip 2.

TABLE 6 Sequencing Results Spot Error rate Cycle efficiency 1 1/763 bp99.87% 2 1/824 bp 99.88% 3 1/780 bp 99.87% 4 1/429 bp 99.77% 5 1/1525bp  99.93% 6 1/1615 bp  99.94% 7 1/531 bp 99.81% 8 1/1769 bp  99.94% 91/854 bp 99.88% 10 1/1451 bp  99.93%

Thus, the high quality and uniformity of the synthesizedoligonucleotides were repeated on two chips with different surfacechemistries. Overall, 89%, corresponding to 233 out of 262 of the100-mers that were sequenced were perfect sequences with no errors.

Finally, Table 7 summarizes error characteristics for the sequencesobtained from the oligonucleotides samples from spots 1-10.

TABLE 7 Error Characteristics Sample ID/Spot no. OSA_0046/1 OSA_0047/2OSA_0048/3 OSA_0049/4 OSA_0050/5 Total 32 32 32 32 32 SequencesSequencing 25 of 28 27 of 27 26 of 30 21 of 23 25 of 26 Quality OligoQuality 23 of 25 25 of 27 22 of 26 18 of 21 24 of 25 ROI Match 2500 26982561 2122 2499 Count ROI 2 2 1 3 1 Mutation ROI Multi 0 0 0 0 0 BaseDeletion ROI Small 1 0 0 0 0 Insertion ROI Single 0 0 0 0 0 BaseDeletion Large 0 0 1 0 0 Deletion Count Mutation: 2 2 1 2 1 G > AMutation: 0 0 0 1 0 T > C ROI Error 3 2 2 3 1 Count ROI Error Err: ~1Err: ~1 Err: ~1 Err: ~1 Err: ~1 Rate in 834 in 1350 in 1282 in 708 in2500 ROI Minus MP Err: ~1 MP Err: ~1 MP Err: ~1 MP Err: ~1 MP Err: ~1Primer in 763 in 824 in 780 in 429 in 1525 Error Rate Sample ID/Spot no.OSA_0051/6 OSA_0052/7 OSA_0053/8 OSA_0054/9 OSA_0055/10 Total 32 32 3232 32 Sequences Sequencing 29 of 30 27 of 31 29 of 31 28 of 29 25 of 28Quality Oligo Quality 25 of 29 22 of 27 28 of 29 26 of 28 20 of 25 ROIMatch 2666 2625 2899 2798 2348 Count ROI 0 2 1 2 1 Mutation ROI Multi 00 0 0 0 Base Deletion ROI Small 0 0 0 0 0 Insertion ROI Single 0 0 0 0 0Base Deletion Large 1 1 0 0 0 Deletion Count Mutation: 0 2 1 2 1 G > AMutation: 0 0 0 0 0 T > C ROI Error 1 3 1 2 1 Count ROI Error Err: ~1Err: ~1 Err: ~1 Err: ~1 Err: ~1 Rate in 2667 in 876 in 2900 in 1400 in2349 ROI Minus MP Err: ~1 MP Err: ~1 MP Err: ~1 MP Err: ~1 MP Err: ~1Primer in 1615 in 531 in 1769 in 854 in 1451 Error Rate

Example 4: Generation of a Nucleic Acid Library by Single-Site, SinglePosition Mutagenesis

Oligonucleotide primers were de novo synthesized for use in a series ofPCR reactions to generate a library of oligonucleotide variants of atemplate nucleic acid, see FIGS. 2A-2D. Four types of primers weregenerated in FIG. 2A: an outer 5′ primer 215, an outer 3′ primer 230, aninner 5′ primer 225, and an inner 3′ primer 220. The inner 5′primer/first oligonucleotide 220 and an inner 3′ primer/secondoligonucleotide 225 were generated using an oligonucleotide synthesismethod as generally outlined in Table 6. The inner 5′ primer/firstoligonucleotide 220 represents a set of up to 19 primers ofpredetermined sequence, where each primer in the set differs fromanother at a single codon, in a single site of the sequence.

Oligonucleotide synthesis was performed on a device having at least twoclusters, each cluster having 121 individually addressable loci.

The inner 5′ primer 225 and the inner 3′ primer 220 were synthesized inseparate clusters. The inner 5′ primer 225 was replicated 121 times,extending on 121 loci within a single cluster. For inner 3′ primer 220,each of the 19 primers of variant sequences were each extended on 6different loci, resulting in the extension of 114 oligonucleotides on114 different loci.

Synthesized oligonucleotides were cleaved from the surface of the deviceand transferred to a plastic vial. A first PCR reaction was performed,using fragments of the long nucleic acid sequence 235, 240 to amplifythe template nucleic acid, as illustrated in FIG. 2B. A second PCRreaction was performed using primer combination and the products of thefirst PCR reaction as a template, as illustrated in FIGS. 2C-2D.Analysis of the second PCR products was conducted on a BioAnalyzer, asshown in the trace of FIG. 17.

Example 5: Generation of a Nucleic Acid Library Comprising 96 DifferentSets of Single Position Variants

Four sets of primers, as generally shown in FIG. 2A and addressed inExample 2, were generated using de novo oligonucleotide synthesis. Forthe inner 5′ primer 220, 96 different sets of primers were generated,each set of primers targeting a different single codon positioned withina single site of the template nucleic acid. For each set of primers, 19different variants were generated, each variant comprising a codonencoding for a different amino acid at the single site. Two rounds ofPCR were performed using the generated primers, as generally shown inFIGS. 2A-2D and described in Example 2. The 96 sets of amplificationproducts were visualized in an electropherogram (FIG. 18), which wasused to calculate a 100% amplification success rate.

Example 6: Generation of a Nucleic Acid Library Comprising 500 DifferentSets of Single Position Variants

Four sets of primers, as generally shown in FIG. 2A and addressed inExample 2, were generated using de novo oligonucleotide synthesis. Forthe inner 5′ primer 220, 500 different sets of primers were generated,each set of primers targeting a different single codon positioned withina single site of the template nucleic acid. For each set of primers, 19different variants were generated, each variant comprising a codonencoding for a different amino acid at the single site. Two rounds ofPCR were performed using the generated primers, as generally shown inFIG. 2A and described in Example 2. Electropherograms display each ofthe 500 sets of PCR products having a population of nucleic acids with19 variants at a different single site (data not shown). A comprehensivesequencing analysis of the library showed a greater than 99% successrate across preselected codon mutations (sequence trace and analysisdata not shown).

Example 7: Single-Site Mutagenesis Primers For 1 Position

An example of codon variation design is provided in Table 8 for YellowFluorescent Protein. In this case, a single codon from a 50-mer of thesequence is varied 19 times. Variant nucleic acid sequence is indicatedby bold letters. The wild type primer sequence is:

(SEQ ID NO.: 1) ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCAT.In this case, the wild type codon encodes for valine, indicated byunderline in SEQ ID NO.: 1. Therefore the 19 variants below exclude acodon encoding for valine. In an alternative example, if all tripletsare to be considered, then all 60 variants would be generated, includingan alternative sequence for the wild type codon.

TABLE 8 Variant Sequences SEQ ID Variant NO. Variant sequence codon 2atgTTTAGCAAGGGCGAGGAGC F TGTTCACCGGGGTGGTGCCCAT 3 atgTTAAGCAAGGGCGAGGAGCL TGTTCACCGGGGTGGTGCCCAT 4 atgATTAGCAAGGGCGAGGAGC ITGTTCACCGGGGTGGTGCCCAT 5 atgTCTAGCAAGGGCGAGGAGC S TGTTCACCGGGGTGGTGCCCAT6 atgCCTAGCAAGGGCGAGGAGC P TGTTCACCGGGGTGGTGCCCAT 7atgACTAGCAAGGGCGAGGAGC T TGTTCACCGGGGTGGTGCCCAT 8 atgGCTAGCAAGGGCGAGGAGCA TGTTCACCGGGGTGGTGCCCAT 9 atgTATAGCAAGGGCGAGGAGC YTGTTCACCGGGGTGGTGCCCAT 10 atgCATAGCAAGGGCGAGGAGC HTGTTCACCGGGGTGGTGCCCAT 11 atgCAAAGCAAGGGCGAGGAGC QTGTTCACCGGGGTGGTGCCCAT 12 atgAATAGCAAGGGCGAGGAGC NTGTTCACCGGGGTGGTGCCCAT 13 atgAAAAGCAAGGGCGAGGAGC KTGTTCACCGGGGTGGTGCCCAT 14 atgGATAGCAAGGGCGAGGAGC DTGTTCACCGGGGTGGTGCCCAT 15 atgGAAAGCAAGGGCGAGGAGC ETGTTCACCGGGGTGGTGCCCAT 16 atgTGTAGCAAGGGCGAGGAGC CTGTTCACCGGGGTGGTGCCCAT 17 atgTGGAGCAAGGGCGAGGAGC WTGTTCACCGGGGTGGTGCCCAT 18 atgCGTAGCAAGGGCGAGGAGC RTGTTCACCGGGGTGGTGCCCAT 19 atgGGTAGCAAGGGCGAGGAGC GTGTTCACCGGGGTGGTGCCCAT

Example 8: Single Site, Dual Position Nucleic Acid Variants

De novo oligonucleotide synthesis was performed under conditions similarto those described in Example 2. A single cluster on a device wasgenerated which contained synthesized predetermined variants of anucleic acid for 2 consecutive codon positions at a single site, eachposition being a codon encoding for an amino acid. In this arrangement,19 variants/per position were generated for 2 positions with 3replicates of each nucleic acid, resulting in 114 nucleic acidssynthesized.

Example 9: Multiple Site, dual Position Nucleic Acid Variants

De novo oligonucleotide synthesis was performed under conditions similarto those described in Example 2. A single cluster on a device wasgenerated which contained synthesized predetermined variants of anucleic acid for 2 non-consecutive codon positions, each position beinga codon encoding for an amino acid. In this arrangement, 19 variants/perposition were generated for 2 positions.

Example 10: Single Stretch, triple Position Nucleic Acid Variants

De novo oligonucleotide synthesis was performed under conditions similarto those described in Example 2. A single cluster on a device wasgenerated which contained synthesized predetermined variants of areference nucleic acid for 3 consecutive codon positions. In the 3consecutive codon position arrangement, 19 variants/per position weregenerated for 3 positions with 2 replicates of each nucleic acid, andresulted in 114 nucleic acids synthesized.

Example 11: Multiple Site, triple Position Nucleic Acid Variants

De novo oligonucleotide synthesis was performed under conditions similarto those described in Example 2. A single cluster on a device wasgenerated which contains synthesized predetermined variants of areference nucleic acid for at least 3 non-consecutive codon positions.Within a predetermined region, the location of codons encoding for 3histidine residues were varied.

Example 12: Multiple Site, multiple Position Nucleic Acid Variants

De novo oligonucleotide synthesis was performed under conditions similarto those described in Example 2. A single cluster on a device wasgenerated which contained synthesized predetermined variants of areference nucleic acid for 1 or more codon positions in 1 or morestretches. Five positions were varied in the library. The first positionencoded codons for a resultant 50/50 K/R ratio in the expressed protein;the second position encoded codons for a resultant 50/25/25 V/L/S ratioin the expressed protein, the third position encoded codons for aresultant a 50/25/25 Y/R/D ratio in the expressed protein, the fourthposition encoded codons for a resultant an equal ratio for all aminoacids in the expressed protein, and the fifth position encoded codonsfor a resultant a 75/25 G/P ratio in the expressed protein.

Example 13: Modular Plasmid Components For Expressing Diverse Peptides

A nucleic acid library is generated as in Examples 4-6 and 8-12,encoding for codon variation at a single site or multiple sites for eachof separate regions that make up portions of an expression constructcassette, as depicted in FIG. 11. To generate a two construct expressingcassette, variant oligonucleotides were synthesized encoding at least aportion of a variant sequence of a first promoter 1110, first openreading frame 1120, first terminator 1130, second promoter 1140, secondopen reading frame 1150, or second terminator sequence 1160. Afterrounds of amplification, as described in previous examples, a library of1,024 expression constructs is generated.

Example 14: Multiple Site, Single Position Variants

A nucleic acid library is generated as in Examples 4-6 and 8-12,encoding for codon variation at a single site or multiple sites in aregion encoding for at least a portion of nucleic acid. A library ofoligonucleotide variants is generated, wherein the library consists ofmultiple site, single position variants. See, for example, FIG. 6B.

Example 15: Variant Library Synthesis

De novo oligonucleotide synthesis is performed under conditions similarto those described in Example 2. At least 30,000 non-identicaloligonucleotides are de novo synthesized, wherein each of thenon-identical oligonucleotides encodes for a different codon variant ofan amino acid sequence. The synthesized at least 30,000 non-identicaloligonucleotides have an aggregate error rate of less than 1 in 1:000bases compared to predetermined sequences for each of the at least30,000 non-identical oligonucleotides. The library is used for PCRmutagenesis of a long nucleic acid and at least 30,000 non-identicalvariant nucleic acids are formed.

Example 16: Cluster-Based Variant Library Synthesis

De novo oligonucleotide synthesis is performed under conditions similarto those described in Example 2. A single cluster on a device isgenerated which contained synthesized predetermined variants of areference oligonucleotide for 2 codon positions. In the 2 consecutivecodon position arrangement, 19 variants/per position were generated forthe 2 positions with 2 replicates of each oligonucleotide, and resultedin 38 oligonucleotides synthesized. Each variant sequence is 40 bases inlength. In the same cluster, additional non-variant oligonucleotidesequences are generated, where the additional non-variantoligonucleotides and the variant oligonucleotides collective encode for38 variants of the coding sequence of a gene. Each of theoligonucleotides has at least one region reverse complementary toanother of the oligonucleotides. The oligonucleotides in the cluster arereleased by gaseous ammonia cleavage. A pin comprising water contactsthe cluster, picks up the oligonucleotides, and moves theoligonucleotides to a small vial. The vial also contains DNA polymerasereagents for a polymerase cycling assembly (PCA) reaction. Theoligonucleotides anneal, gaps are filled in by an extension reaction,and resultant double-stranded DNA molecules are formed, forming avariant nucleic acid library. The variant nucleic acid library is,optionally, subjected to restriction enzyme is then ligated intoexpression vectors.

Example 17: Generation of a Variant Nucleic Acid Library

CAR variant libraries are generated, as described in Examples 8-16, tohave variance in an antigen recognition domain, a hinge domain, atransmembrane domain, or an intracellular domain compared to a referencesequence. Alternatively, CAR variant libraries are also generated tohave variance in all domains of a CAR. The CAR variant libraries arescreened against a known tumor antigen. Variant sequences that result inincreased T cell activation and/or T cell binding are identified. Thetumor antigen may be expressed on a primary cultured cell, non-primarycultured cell, or in a non-cellular setting, such as on a plate, beads,slurry, or column.

Example 18: Expression and Screening of a Variant CAR Library

The library of variant CAR genes described in Example 17 are transferredinto mammalian cells to generate a library of cell populations, eachcell population expressing a different CAR variant protein. The proteinlibrary is screened for CAR complexes with improved affinity (measure ofthe strength of interaction between an epitope and an antibody's antigenbinding site) for a tumor antigen, such as HER2 or NY-ESO-1. Additionalfunctional considerations, such as variant gene expression, avidity(measure of the overall strength of an antibody-antigen complex),stability, and target specificity are also assessed.

Example 19: Manufacturing and Delivery of Engineered T Cells

T cells are harvested from a subject diagnosed with cancer and aregenetically engineered with a CAR selected after performing analysis inExample 18. After a brief period of in vitro expansion and passing ofproduct-specific release criteria, the engineered T-cells areadministered to the same subject.

Example 20: Variant Peptide Library

A nucleic acid library is generated as in Examples 4-6 and 8-12,encoding for codon variation at a single site or multiple sites forcommon tumor antigens. After rounds of amplification, as described inprevious examples, libraries of 1,024 expression constructs for each ofthe antigen variants are generated. The libraries are transferred intomammalian cells to generate a library of cell populations and arescreened for improved binding affinity and target specificity.

Example 21: Variant Peptide Library and Variant Library of CARs

A nucleic acid is generated as in Examples 4-6 and 8-12, encoding forcodon variation at a single site or multiple sites for an antigenrecognition domain of a CAR. A nucleic acid library is generated as inExample 20, encoding for codon variation at a single site or multiplesites for common tumor antigens. After rounds of amplification, asdescribed in previous examples, libraries of 1,024 expression constructsfor each of the CAR variants and the antigen variants are generated. Thelibraries are transferred into mammalian cells to generate a library ofcell populations and are screened for improved binding affinity andtarget specificity.

Example 22: Variant CARs

A nucleic acid library is generated as in Examples 4-6 and 8-12 togenerate nucleic acids encoding for variant CARs. Variants are generatedat three sites to generate a library comprising about 10³ variant CARs.

The variant CARs are screened in vitro against tumor antigens forspecificity of the variant CARs to the tumor antigen. Variant CARs thatare highly specific for the tumor antigen are then further variegated togenerate a second library. The second library comprises variation inresidues located in the hinge domain, the transmembrane domain, or theintracellular domain of the CAR. The second library is expressed incells and screened in vitro for a second improvement, for example,avidity, stability, affinity, or expression.

Select CAR genes or gene fragment variants having desired features afterscreening products from the first and/or second variant libraries areselected for development of a potential therapeutic.

While preferred embodiments of the present disclosure have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the disclosure. It should beunderstood that various alternatives to the embodiments of thedisclosure described herein may be employed in practicing thedisclosure. It is intended that the following claims define the scope ofthe disclosure and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

What is claimed is:
 1. A nucleic acid library, the nucleic acid library comprising at least 10,000 nucleic acids, wherein each nucleic acid encodes for a preselected variant of a reference sequence that encodes for a chimeric antigen receptor or a functional domain thereof.
 2. The nucleic acid library of claim 1, wherein the functional domain of the chimeric antigen receptor comprises an antigen recognition domain, a hinge domain, a transmembrane domain, or an intracellular domain.
 3. The nucleic acid library of claim 1, wherein the library comprises at least 1,000,000 nucleic acids.
 4. The nucleic acid library of claim 1, wherein each nucleic acid is 100 bases to 2000 bases in length.
 5. The nucleic acid library of claim 1, wherein each nucleic acid is attached to a vector sequence.
 6. The nucleic acid library of claim 5, wherein the vector sequence is a viral vector sequence.
 7. A nucleic acid library, the nucleic acid library comprising a plurality of nucleic acids, wherein each nucleic acid encodes for a preselected variant of a reference sequence that encodes for a chimeric antigen receptor or a functional domain thereof, wherein the plurality of nucleic acids comprises all variations for at least two positions in the reference sequence, and wherein the at least two positions encode sequences from 5 to 20 different codons.
 8. The nucleic acid library of claim 7, wherein the functional domain of the chimeric antigen receptor comprises an antigen recognition domain, a hinge domain, a transmembrane domain, or an intracellular domain.
 9. The nucleic acid library of claim 7, wherein the plurality of nucleic acids comprises all variations for 2, 3, 4, or 5 positions in the reference sequence.
 10. The nucleic acid library of claim 7, wherein the at least two positions encode sequences from 10 to 20 different codons.
 11. The nucleic acid library of claim 7, wherein the at least two positions encode sequences for about 10 different codons.
 12. The nucleic acid library of claim 7, wherein each nucleic acid is 100 bases to 2000 bases in length.
 13. An oligonucleotide library, the oligonucleotide library comprising at least 10,000 oligonucleotides, wherein each oligonucleotide encodes for a preselected variant of a reference sequence that encodes for a region of a chimeric antigen receptor, wherein the region comprises a portion of an antigen recognition domain, a hinge domain, a transmembrane domain, or an intracellular domain.
 14. The oligonucleotide library of claim 13, wherein the library comprises at least 1,000,000 oligonucleotides.
 15. The oligonucleotide library of claim 13, wherein each oligonucleotide is 12 to 500 bases in length.
 16. The oligonucleotide library of claim 13, wherein each oligonucleotide is attached to a vector sequence.
 17. An oligonucleotide library, the oligonucleotide library comprising a plurality of oligonucleotides, wherein each oligonucleotide encodes for a preselected variant of a reference sequence that encodes for a region of a chimeric antigen receptor, wherein the region comprises a portion of an antigen recognition domain, a hinge domain, a transmembrane domain, or an intracellular domain, wherein each oligonucleotide is at least 12 bases in length, wherein the plurality of oligonucleotides comprises all variations for at least two positions in the reference sequence, and wherein the at least two positions encode sequences from 5 to 20 different codons.
 18. The oligonucleotide library of claim 17, wherein the plurality of oligonucleotides comprises all variations for 2, 3, 4, or 5 positions.
 19. The oligonucleotide library of claim 17, wherein the at least two positions encode sequences from 10 to 20 different codons.
 20. The oligonucleotide library of claim 17, wherein the at least two positions encode sequences for about 10 different codons.
 21. The oligonucleotide library of claim 17, wherein each oligonucleotide is 12 to 500 bases in length.
 22. A nucleic acid library comprising at least 400 nucleic acids, wherein a first plurality of the at least 400 nucleic acids encodes for a variant of a reference sequence that encodes for an antigen recognition domain, and wherein a second plurality of the at least 400 nucleic acids encodes for a variant of a reference sequence that encodes for a hinge domain, a transmembrane domain, or an intracellular domain of a chimeric antigen receptor (CAR).
 23. The nucleic acid library of claim 22, wherein each nucleic acid is at least 100 bases in length.
 24. The nucleic acid library of claim 22, wherein each nucleic acid is 100 to 2000 bases in length.
 25. The nucleic acid library of claim 22, wherein the first plurality of the at least 400 nucleic acids comprises all variations for at least two positions in the reference sequence.
 26. The nucleic acid library of claim 22, wherein the first plurality of the at least 400 nucleic acids comprises all variations for 2, 3, 4, or 5 positions in the reference sequence.
 27. The nucleic acid library of claim 22, wherein the second plurality of the at least 400 nucleic acids comprises all variations for at least two positions in the reference sequence.
 28. The nucleic acid library of claim 22, wherein the second plurality of the at least 400 nucleic acids comprises all variations for 2, 3, 4, or 5 positions in the reference sequence.
 29. The nucleic acid library of claim 25, wherein the at least two positions encode sequences from 5 to 20 different codons.
 30. The nucleic acid library of claim 25, wherein the at least two positions encode sequences from 10 to 20 different codons.
 31. The nucleic acid library of claim 25, wherein the at least two positions encode sequences for about 10 different codons. 